U.S. patent application number 14/580680 was filed with the patent office on 2015-06-25 for rac1 inhibitors for the treatment of alport glomerular disease.
The applicant listed for this patent is Father Flanagan's Boys' Home doing business as Boys Town National Research Hospital. Invention is credited to Dominic Cosgrove.
Application Number | 20150175695 14/580680 |
Document ID | / |
Family ID | 53399301 |
Filed Date | 2015-06-25 |
United States Patent
Application |
20150175695 |
Kind Code |
A1 |
Cosgrove; Dominic |
June 25, 2015 |
RAC1 INHIBITORS FOR THE TREATMENT OF ALPORT GLOMERULAR DISEASE
Abstract
The present invention provides methods of treating Alport
syndrome in a subject by the administration of an agent that can
blocks the activation of RAC1/CDC42 members of the rho family of
small GTPases. Such agents include, but are not limited to, the
endothelin receptor antagonists such as bosentan and letairis and
neutralizing antibodies to endothelin-1. Such administration
prevents invasion of the glomerular capillary tufts by mesangial
lamellipodial/filopodial processes, blocks mesangial process
invasion abrogates the deposition of laminin 211 in the GBM, and
prevents the activation of maladaptive expression of proteins known
to contribute to glomerular disease progression.
Inventors: |
Cosgrove; Dominic; (Omaha,
NE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Father Flanagan's Boys' Home doing business as Boys Town National
Research Hospital |
Omaha |
NE |
US |
|
|
Family ID: |
53399301 |
Appl. No.: |
14/580680 |
Filed: |
December 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2013/032432 |
Mar 15, 2013 |
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14580680 |
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61684566 |
Aug 17, 2012 |
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61764389 |
Feb 13, 2013 |
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61920055 |
Dec 23, 2013 |
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62079988 |
Nov 14, 2014 |
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Current U.S.
Class: |
424/172.1 ;
435/29; 514/235.8; 514/269; 514/274; 514/275 |
Current CPC
Class: |
A61K 31/5377 20130101;
G01N 33/5044 20130101; G01N 33/5026 20130101; A61K 39/395 20130101;
G01N 2800/50 20130101; A61K 31/4025 20130101; G01N 2800/347
20130101; G01N 2500/10 20130101; A61K 31/506 20130101; A61K 31/505
20130101; G01N 33/6893 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/50 20060101 G01N033/50; A61K 31/5377 20060101
A61K031/5377; C07K 16/18 20060101 C07K016/18; A61K 31/506 20060101
A61K031/506; A61K 31/505 20060101 A61K031/505 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under Grant
No. R01-DK55000 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method of inhibiting Alport pathogenesis in a subject; the
method comprising administering an effective amount of a RAC1
inhibitor and/or a CDC42 inhibitor to the subject.
2. The method of claim 1, wherein glomerular disease progression is
inhibited.
3. The method of claim 1, wherein glomerulonephritis is
treated.
4. The method of claim 1, wherein deposition of laminin 211 in the
glomerular basement membrane (GBM) is inhibited.
5. The method of claim 1, wherein mesangial cell process invasion
of the glomerular capillary loop in a kidney is inhibited.
6. The method of claim 1, wherein one or more sensory and/or
hearing losses associated with Alport syndrome is treated or
prevented.
7. A method of inhibiting Alport glomerular pathogenesis in a
subject; the method comprising: determining that the subject is at
risk for developing Alport glomerular disease; and administering an
effective amount of a RAC1 inhibitor and/or a CDC42 inhibitor to
the subject.
8. The method of claim 7, wherein the determination that the
subject is at risk for developing Alport glomerular disease is
determined by family medical history, genetic testing,
immunodiagnostic skin biopsy testing, and/or molecular diagnostic
marker testing.
9. The method of claim 7, wherein the determination that the
subject is at risk for developing Alport glomerular disease is made
prior to the onset of proteinuria in the subject.
10. The method of claim 1, wherein the administration of an
effective amount of a RAC1 inhibitor and/or a CDC42 inhibitor is
initiated prior to the onset of proteinuria in the subject.
11. The method of claim 1, wherein the RAC1 inhibitor and/or a
CDC42 inhibitor is an agent that blocks activation of the
endothelin type I receptor and/or the endothelin type II
receptor.
12. The method of claim 1, wherein the RAC1 inhibitor and/or a
CDC42 inhibitor comprises an endothelin (ET) receptor
antagonist.
13. The method of claim 12, wherein the endothelin (ET) receptor
antagonist comprises a dual antagonist of both the ET.sub.A
receptor and ET.sub.B receptor.
14. The method of claim 12, wherein the endothelin (ET) receptor
antagonist comprises an antagonist of the ET.sub.A receptor.
15. The method of claim 1, wherein the RAC1 inhibitor and/or CDC42
inhibitor comprises bosentan (TRACLEER.RTM.), ambrisentan
(LETAIRIS.RTM.), NSC23766, TAE226, macitentan (OPSUMIT.RTM.),
altrasentan, or a derivative thereof.
16. The method of claim 1, wherein the RAC1 and/or CDC42 inhibitor
comprises an antibody against endothelin-1 or a derivative
thereof.
17. The method of claim 16, wherein the antibody against
endothelin-1 or derivative thereof neutralizes one or more
functions of endothelin-1.
18. The method of claim 1, wherein the RAC1 and/or CDC42 inhibitor
comprises an antibody against endothelin A receptor or a derivative
thereof.
19. The method of claim 18, wherein the antibody against endothelin
A receptor or derivative thereof neutralizes one or more functions
of endothelin A receptor.
20. An in vitro method of identifying an endothelin receptor
antagonist agent effective for treating Alport syndrome in a
subject, preventing glomerular disease progression in a subject
diagnosed with Alport syndrome, treating glomerulonephritis in a
subject, treating kidney injury due to biomechanical strain in
Alport syndrome, inhibiting deposition of laminin 211 in the
glomerular basement membrane (GBM) in a subject, inhibiting
mesangial cell process invasion of the glomerular capillary loop in
a kidney of a subject, inhibiting Alport glomerular pathogenesis in
a subject, and/or treating or preventing one or more of the eye or
hearing pathologies associated with Alport syndrome in a subject,
the method comprising: contacting cultured mesangial cells or
cultured podocytes with the agent; contacting the cultured cells
with endothelin-1; observing the formation of drebrin-positive
filopodial microspikes; wherein an effective agent inhibits,
reduces, and/or blocks the formation of drebrin-positive filopodial
microspikes.
Description
CONTINUING APPLICATION DATA
[0001] This application is a continuation-in-part of International
Application No. PCT/US2013/032432, filed Mar. 15, 2013, which
claims the benefit of U.S. Provisional Application Ser. No.
61/684,566, filed Aug. 17, 2012, and U.S. Provisional Application
Ser. No. 61/764,389, filed Feb. 13, 2013, all of which is
incorporated by reference herein. This application also claims
priority to U.S. Provisional Application No. 61/920,055, filed Dec.
23, 2013, and U.S. Provisional Application No. 62/079,988, filed
Nov. 14, 2014, each of which is incorporated by reference
herein.
BACKGROUND
[0003] Alport syndrome (also referred to as hereditary nephritis)
is a genetic disorder characterized by abnormalities in the
basement membranes of the glomerulus (leading to hematuria,
glomerulosclerosis, and end-stage kidney disease (ESRD)), cochlea
(causing deafness), and eye (resulting in lenticonus and
perimacular flecks). Alport syndrome is a primary basement membrane
disorder caused by mutations in the collagen type IV COL4A3,
COL4A4, or COL4A5 genes. Mutations in any of these genes prevent
the proper production or assembly of the type IV collagen network,
which is an important structural component of basement membranes in
the kidney, inner ear, and eye. Basement membranes are thin,
sheet-like structures that separate and support cells in many
tissues. The abnormalities of type IV collagen in kidney glomerular
basement membranes leads to irregular thickening and thinning and
splitting of these basement membranes, causing gradual scarring
(fibrosis) of the kidneys. Alport Syndrome has a delayed onset and
causes progressive kidney damage. The glomeruli and other normal
kidney structures such as tubules are gradually replaced by scar
tissue, leading to kidney failure. Hearing loss and an abnormality
in the shape of the lens called anterior lenticonus are other
important features of Alport Syndrome. People with anterior
lenticonus may have problems with their vision and may develop
cataracts. The prevalence of Alport syndrome is estimated at
approximately 1 in 5,000 births and it is estimated that the
syndrome accounts for approximately 2.1 percent of pediatric
patients with ESRD. Currently there is no specific treatment for
Alport Syndrome; treatments are symptomatic. Patients are advised
on how to manage the complications of kidney failure and the
proteinuria that develops is often treated with ACE inhibitors.
Once kidney failure has developed, patients are given dialysis or
can benefit from a kidney transplant, although this can cause
problems. The body may reject the new kidney as it contains normal
type IV collagen, which may be recognized as foreign by the immune
system. Thus there is a need for improved therapeutic approaches
for the treatment of Alport syndrome.
SUMMARY OF INVENTION
[0004] The present invention includes a method of treating Alport
syndrome in a subject, the method including administering an
effective amount of a RAC1 inhibitor and/or a CDC42 inhibitor.
[0005] The present invention includes a method of preventing
glomerular disease progression in a subject diagnosed with Alport
syndrome, the method including administering an effective amount of
a RAC1 inhibitor and/or a CDC42 inhibitor.
[0006] The present invention includes a method of treating
glomerulonephritis in a subject, the method including administering
an effective amount of a RAC1 inhibitor and/or a CDC42
inhibitor.
[0007] The present invention includes a method of treating kidney
injury due to biomechanical strain in Alport syndrome, the method
including administering an effective amount of a RAC1 inhibitor
and/or a CDC42 inhibitor.
[0008] The present invention includes a method of inhibiting
deposition of laminin 211 in the glomerular basement membrane (GBM)
in a subject, the method including administering an effective
amount of a RAC1 inhibitor and/or a CDC42 inhibitor.
[0009] The present invention includes a method of inhibiting
mesangial cell process invasion of the glomerular capillary loops
in a kidney of a subject, the method including administering an
effective amount of a RAC1 inhibitor and/or a CDC42 inhibitor.
[0010] The present invention includes a method of inhibiting Alport
glomerular pathogenesis in a subject; the method including:
determining that the subject is at risk for developing Alport
glomerular disease; and administering an effective amount of a RAC1
inhibitor and/or a CDC42 inhibitor to the subject. In some aspects,
the determination that the subject is at risk for developing Alport
glomerular disease is determined by family medical history, genetic
testing, immunodiagnostic skin biopsy testing, and/or molecular
diagnostic marker testing. In some aspects, the determination that
the subject is at risk for developing Alport glomerular disease is
made prior to the onset of proteinuria in the subject.
[0011] The present invention includes a method of treating or
preventing one or more aspects of a sensory loss and/or hearing
loss associated with Alport syndrome in a subject, the method
including administering an effective amount of a RAC1 inhibitor
and/or a CDC42 inhibitor.
[0012] In some aspects of the methods of the present invention, the
administration of an effective amount of a RAC1 inhibitor and/or a
CDC42 inhibitor is initiated prior to the onset of proteinuria in
the subject.
[0013] In some aspects of the methods of the present invention, the
RAC1 inhibitor and/or a CDC42 inhibitor is an agent that blocks
activation of the endothelin type I receptor and/or the endothelin
type II receptor.
[0014] In some aspects of the methods of the present invention, the
RAC1 inhibitor and/or a CDC42 inhibitor is an endothelin (ET)
receptor antagonist. In some aspects, the endothelin (ET) receptor
antagonist is a dual antagonist of both the ET.sub.A receptor and
ET.sub.B receptor. In some aspects, the endothelin (ET) receptor
antagonist is an antagonist of the ET.sub.A receptor. In some
aspects, the endothelin (ET) receptor antagonist is an antagonist
of the ET.sub.B receptor. In some aspects of the methods of the
present invention, the endothelin (ET) receptor antagonist is
bosentan or a derivative thereof.
[0015] In some aspects of the methods of the present invention, the
RAC1 inhibitor and/or a CDC42 inhibitor is bosentan or a derivative
thereof. In some aspects of the methods of the present invention,
the RAC1 inhibitor and/or a CDC42 inhibitor is letairius or a
derivative thereof. In some aspects of the methods of the present
invention, the RAC1 inhibitor is NSC23766 or a derivative
thereof.
[0016] In some aspects of the methods of the present invention, the
endothelin (ET) receptor antagonist is letairius or a derivative
thereof.
[0017] In some aspects of the methods of the present invention, the
endothelin (ET) receptor antagonist is NSC23766 or a derivative
thereof.
[0018] In some aspects of the methods of the present invention, the
RAC1 and/or CDC42 inhibitor includes macitentan (OPSUMIT.RTM.) or a
derivative thereof.
[0019] In some aspects of the methods of the present invention, the
RAC1 and/or CDC42 inhibitor includes altrasentan or a derivative
thereof.
[0020] In some aspects of the methods of the present invention, the
RAC1 and/or CDC42 inhibitor includes an antibody that specifically
binds to endothelin-1 or a derivative thereof. In some aspects, the
antibody against endothelin-1 or derivative thereof neutralizes one
or more functions of endothelin-1.
[0021] In some aspects of the methods of the present invention, the
RAC1 and/or CDC42 inhibitor includes an antibody that specifically
binds an endothelin receptor or a derivative thereof. In some
aspects, the antibody against an endothelin receptor or derivative
thereof neutralizes one or more functions of an endothelin
receptor.
[0022] In some aspects of the methods of the present invention, the
RAC1 and/or CDC42 inhibitor includes an antibody that specifically
binds the endothelin A receptor or a derivative thereof. In some
aspects, the antibody against endothelin A receptor or derivative
thereof neutralizes one or more functions of endothelin A
receptor.
[0023] The present invention also includes an in vitro bioassay for
identifying agents effective for treating Alport syndrome in a
subject, preventing glomerular disease progression in a subject
diagnosed with Alport syndrome, treating glomerulonephritis in a
subject, treating kidney injury due to biomechanical strain in
Alport syndrome, inhibiting deposition of laminin 211 in the
glomerular basement membrane (GBM) in a subject, inhibiting
mesangial cell process invasion of the glomerular capillary loop in
a kidney of a subject, and/or inhibiting Alport glomerular
pathogenesis in a subject.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1. Laminin 211 localizes to the glomerular basement
membrane (GBM) in Alport glomeruli. Dual immunofluorescence
immunostaining was performed on wild type (A-C) and Alport (D-F)
glomeruli from 7 week 129 Sv mice. Glomerular basement membranes
were labeled with labeled with anti-laminin .alpha.5 antibodies (A
and D). Anti-laminin .alpha.2 immunostaining is shown in B and E.
Note the irregular deposits of laminin 211 in the Alport GBM,
especially in the thickened regions of the GBM (overlapping
staining in D and E). Anti-laminin .alpha.2 immunostaining is not
observed in the GBM of wild type mice (note the absence of
overlapping immunostaining in A and B).
[0025] FIG. 2. Mesangial processes invade the capillary loops of
Alport glomeruli where they co-localize with laminin 211 deposits.
Dual immunofluorescence immunostaining was performed on wild type
or Alport kidney sections from 7 week old 129 Sv mice. A-F show
localization of laminin .alpha.2 and integrin .alpha.8 (a mesangial
cell marker), and G-L FIGS. 2G-2L show localization of laminin
.alpha.5 (a GBM marker) and integrin .alpha.8. Note circumferential
co-localization of laminin .alpha.2 and integrin .alpha.8 in the
Alport glomerulus in D-F, and the co-localization of integrin
.alpha.8 and laminin .alpha.5 in J-L indicating invasion of the
glomerular capillary tufts with mesangial processes.
[0026] FIG. 3. Mesangial processes invade the capillary loops of
human Alport glomeruli where they co-localize with laminin 511.
Cryosections from human Alport kidneys were stained with antibodies
specific for laminin .alpha.5 (A) and integrin .alpha.8 (B). The
integrin .alpha.8-specific mesangial processes localize adjacent to
the laminin .alpha.5-positive GBM, consistent with mesangial
process invasion. C represents a merging of A and B.
[0027] FIG. 4. Hypertension exacerbates mesangial invasion of the
glomerular capillary tufts in Alport mice. The X-linked Alport
mouse model (on the C57 Bl/6 background) was made hypertensive by
providing L-NAME salts in the drinking water from 5 weeks to 10
weeks of age. Control Alport mice were given normal drinking water.
Glomeruli were analyzed by dual immunofluorescence immunostaining
using antibodies against either laminin .alpha.2 (A and D) or
integrin .alpha.8 (B and E). Panels C and F represent a merging of
results with laminin .alpha.2 and integrin .alpha.8 staining.
Extensive mesangial process invasion of the capillary tuft is
observed in the glomeruli from the salt-treated mice relative to
the mice given normal drinking water.
[0028] FIG. 5. Extensive mesangial process invasion of the
glomerular capillary tufts is observed in CD151 knockout mice.
Kidney cryosections from 8 week old wild type and CDC151 KO mice
(on the FVB background) were analyzed by dual immunofluorescence
immunostaining using antibodies against either laminin .alpha.2 or
integrin .alpha.8. Extensive mesangial process invasion of the
capillary tuft is observed in the glomeruli from CD151 knockout
mice relative to wild type mice. Note that the extent of mesangial
process invasion in CD151 knockout mice is much greater than that
observed in Alport mice.
[0029] FIG. 6. Biomechanical stretching of cultured primary
mesangial cells induces expression of pro-migratory cytokines, CTGF
and TGF-.beta.1 mRNA. Primary mesangial cell cultures from wild
type mice were subjected to cyclic biomechanical stretching for 24
hours. RNA from multiple replicates was analyzed by quantitative
real time RT-PCR for CTGF and TGF-.beta.1 mRNA. Statistically
significant increases in expression for both cytokines was observed
(p<0.05).
[0030] FIG. 7. .alpha.1 integrin deletion in Alport mice results in
markedly reduced mesangial process invasion of the glomerular
capillary tufts. Glomeruli from 7 week old integrin .alpha.1-null
mice, Alport mice, and integrin .alpha.1-null Alport mice were
analyzed by dual immunofluorescence immunostaining using antibodies
against either laminin .alpha.2 or integrin .alpha.8. The degree of
mesangial process invasion of the glomerular capillary tufts was
greatly reduced in the integrin .alpha.1-null Alport mice relative
to age/strain-matched Alport mice.
[0031] FIG. 8. Integrin .alpha.1.beta.1-dependent Rac1/CDC42
activation mediates dynamic remodeling of the actin cytoskeleton
and mesangial process invasion of the glomerular capillary tufts.
A) Migration of primary cultured mesangial cells is significantly
reduced under conditions of integrin .alpha.1 deletion, Integrin
linked kinase inhibition, Rac1 inhibition, and CDC42 inhibition,
but not AKT inhibition. In contrast, the migratory potential of
cultured integrin .alpha.1-null mesangial cells is unaffected by
inhibition of either Rac1 or CDC42. Migration was measured by
Boyden chamber assay in the presence or absence of ILK inhibitor,
QLT-0267; Rac1 inhibitor, NSC 23766; CDC42 inhibitor, ML141; or the
pan-AKT inhibitor GSK 690693. Multiple replicate experiments were
performed on multiple independent derivations of mesangial cells
and the data analyzed by Students-t-test. Asterisks denote
statistically significant differences relative to 10% FCS
(p<0.05). B) Treatment of cultured mesangial cells with LPS
induced cytoskeletal rearrangement with numerous actin spikes
(untreated cells, A; LPS treated cells, B), and these morphological
changes are blocked by treatment of cells with either Rac1
inhibitors (C), or CDC42 inhibitors (D). Untreated integrin
.alpha.1-null cells did not respond to LPS treatment. C) Treatment
of cultured mesangial cells with LPS results in polarized
localization of CDC42 and associated with filopodia (B, insert,
compared to Golgi and cytosolic localization of CCD42 in wild type
cells (A). Pre-treatment of cells with the Rac1 inhibitor, NSC
23766, abolished LPS-activated polarized localization of CDC42 (C),
indicating cross-talk between Rac1 and CDC42. D shows a GTP-Rac1
pull down assay which confirms LPS-mediated activation of Rac1 in
cultured mesangial cells, which was blocked by pre-treatment with
Rac1 inhibitors, but not CDC42 inhibitor.
[0032] FIG. 9. Treatment of Alport mice with Rac1 inhibitors
partially ameliorates mesangial cell process invasion of the
glomerular capillary tufts. Alport mice on the 129 Sv background
were injected once daily with either saline or the Rac1 inhibitor
NSC 23766 from 2 weeks to 6 weeks of age. Kidney cryosections were
analyzed by dual immunofluorescence immunostaining using antibodies
against either laminin .alpha.2 (B and E) or integrin .alpha.8 (A
and D). C and D represent a merging of results from staining with
laminin .alpha.2 and integrin .alpha.8. The degree of mesangial
process invasion of the glomerular capillary tufts was ameliorated
in the Rac1 inhibitor-treated mice relative to mice injected with
saline.
[0033] FIG. 10. Laminin 211 potentiates mesangial process invasion
of the glomerular capillary loops in Alport mice, and promotes
mesangial cell migration in vitro. A) Laminin laminin
.alpha.2-deficient Alport mice show reduced mesangial process
invasion of the glomerular capillary tufts. Cryosections of kidney
tissue from 8 week old laminin .alpha.2-deficient Alport mice were
analyzed by dual immunofluorescence immunostaining using antibodies
against either laminin .alpha.5 or integrin .alpha.8. The degree of
mesangial process invasion of the glomerular capillary tufts was
greatly reduced in the laminin .alpha.2-null Alport mice relative
to Alport mice (compare with FIG. 2, panels J-L). B) Wild type
mesangial cells migrate more robustly on laminin 211 compared to
laminin 521 (GBM laminin). Wound scratch assays were performed
using wild type mesangial cells cultured on wither recombinant
purified laminins or commercially available laminins extracted from
either placenta (primarily laminin 511) or muscle (primarily
laminin 211). Images shown are representative of multiple
replicates. C) Primary mesangial cells from laminin
.alpha.2-deficient mice show impaired migratory potential relative
to wild type mesangial cells. Boyden chamber assays were performed.
Blinded cell counts from multiple replicates were analyzed.
Asterisk denotes statistically significant differences
(p<0.05).
[0034] FIG. 11. Hypertension induces endothelin-1 in Alport
glomerular endothelial cells. Wild type and C57Bl/6 X-linked Alport
mice were made hypotensive by giving them the ACE inhibitor
Ramipril in drinking water from 4 to 7 weeks of age, normotensive
by giving plain drinking water, or hypertensive by giving them
L-NAME salts in the drinking water. Cryosections were dual
immunostained with antibodies specific for either endothelin-1 (A,
C, and E) or CD31 (B, D, and F) (a marker for endothelial cells).
ET-1, endothelin-1. Note very little ET-1 immunostaining in the
glomeruli of hypotensive mice (A) versus robust endothelial
cell-specific immunostaining for ET-1 in the glomeruli of
hypertensive mice (E).
[0035] FIG. 12. Inducement of hypertension and hypotension in
Alport mice. X-linked Alport mice on the C57 BL/6 background were
given either Ramipril (angiotensin converting enzyme inhibitor) or
L-NAME salts from the ages of 4 to 7 weeks of age to induce a state
of hypotension or hypertension, respectively. The top graph
represents independent serial blood pressure measurements on 5
animals per group at the indicated ages. Blood pressure
measurements were done using the non-invasive CODA2 tail cuff
system. At 7 weeks this strain of Alport mouse is pre-proteinuric
indicating a state prior to the onset of basement membrane
destruction. The bottom graphs show Bosentan treatment reduces mRNA
expression of MMP-10, MMP-12, MCP-1, and TGF.beta.1 in glomeruli
from Alport mice. Glomerular RNA from Bosentan-treated and
vehicle-treated 129 Sv wild type and Alport mice (once daily from 2
to 7 weeks of age, 100 mg/kg by oral gavage) was analyzed by real
time RT-PCR for the indicated transcripts. MMP, matrix
metalloproteinase; MCP-1, monocyte chemoattractant protein-1;
TGF.beta.1, transforming growth factor beta-1.
[0036] FIG. 13. Bosentan treatment ameliorates interstitial
fibrosis and monocytic infiltration in Alport kidneys. Cryosections
from Bosentan treated and vehicle treated wild type and Alport mice
were immunostained with antibodies specific for fibronectin (A, C,
and E) and CD11b (a monocyte marker) (B, D, and F).
[0037] FIG. 14. Bosentan treatment blocks mesangial process
invasion of Alport glomerular capillaries. Cryosections from wild
type mice and Alport mice given either vehicle or Bosentan were
immunostained using antibodies specific for integrin .alpha.8 (a
mesangial cell surface marker) (A, C, and E) or laminin .alpha.5 (a
marker for the glomerular basement membrane. (B, D, and E).
Co-localization of the two markers (examples of which are denoted
by arrowheads) indicate regions of the GBM infiltrated by mesangial
processes.
[0038] FIG. 15. The endothelin receptor A co-localizes with
integrin .alpha.8, which shows it is abundantly expressed on
mesangial cells in mice.
[0039] FIG. 16. Activation of focal adhesion kinase occurs
specifically in regions of the GBM where laminin .alpha.2 is
present, and is a very early event in Alport glomerular
pathogenesis. Cryosections from 10 day old Alport mice (D-F), 7
week old Alport mice (G-I), and wild type littermates (A-C) were
immunostained with antibodies specific for the .alpha.2 chain of
laminin or pFAK.sup.397. Arrowheads denote areas of dual
immunostaining in the glomerular capillary loops. Scale bar=15
.mu.m.
[0040] FIG. 17. Laminin 211, but not laminin 111 activates FAK on
podocytes in vivo and in vitro. 7 week old wild type glomerulus
stained with antibodies specific for laminin 111 and pFAK.sup.397
show absence of pFAK immunostaining (A-C). 7 week Alport glomerulus
stained with antibodies specific for laminin 111 and pFAK.sup.397
pFAK immunostaining in podocytes adjacent to laminin
111-immunopositive GBM (D-F). G-I show the same immunostaining as
for D-F using Alport mice that do not express laminin 211 (the
dy/dy muscular dystrophy mutation). Note the absence of
pFAK.sup.397 immunostaining even though GBM is immunopositive for
laminin 111. J) Wild type podocytes cultured on merosin (laminin
211) show activated pFAK.sup.397 relative to cells cultured on
placental laminin (laminin 521), or EHS laminin (laminin 111). Wild
type podocytes were differentiated for 2 weeks and then plated on
placental laminin, EHS laminin, or merosin for 15 hours, extracts
were prepared and analyzed by western blot for expression of
pFAK.sup.397 and total FAK. .beta.-actin was used as a loading
control). Scale bar=10 .mu.m.
[0041] FIG. 18. Activation of focal adhesion kinase occurs
specifically in regions of the GBM where laminin .alpha.2 is
present in CD151 knockout mice. Cryosections from 10 week old CD151
knockout mice (D-F) and wild type littermates (A-C) were
immunostained with antibodies specific for the .alpha.2 chain of
laminin or pFAK.sup.397. Arrowheads denote areas of dual
immunostaining along the capillary loops. Scale bar=15 .mu.m.
[0042] FIG. 19. Induction kinetics for MMP-9, MMP-10, MMP-12, IL-6,
and NF-kappaB in glomeruli from Alport mice and CD151 knockout
mice. A) Glomeruli were isolated from CD151 knockout mice and
Alport mice along with strain/age matched wild type littermates at
the indicated ages using magnetic bead isolation. Total glomerular
RNA was analyzed by real time RT-PCR using primers specific for the
indicated transcripts. Each data point represents at least five
independent samples. Significant differences when comparing the
data from mutants with wild type littermates are denoted with
asterisks (p<0.05). Note that IL-6 and NF-kappaB did not reach
significance likely due to a large variance in the data, but
trended towards significance. B) MMP-10 protein is induced in
Alport glomeruli at both 4 and 7 weeks of age as determined by
immunofluorescence analysis. Scale bar=15 .mu.m.
[0043] FIG. 20. Stable siRNA knock-down of FAK in cultured
podocytes results in significantly reduced expression of MMP-9,
MMP-10, and NF-kappaB. Conditionally immortalized podocyte cell
cultures were transfected with vector encoding a siRNA expression
cassette for FAK. A vector encoding a scrambled siRNA was used as a
control. Stable clones were selected and propagated. The data
presented is representative of several independently selected
clones. A-B) While cells expressing the scrambled vector still have
robust focal adhesions (A), they are significantly reduced or
absent in the cells expressing the FAK siRNA (B). C) Western blot
for total FAK confirms a reduction of FAK protein in the FAK siRNA
transfected cultures. D) Real time qRT-PCR analysis of RNA from
these clones shows a significant reduction in the expression of
mRNAs encoding FAK, MMP-9, MMP-10, and NF-kappaBia in FAK siRNA
expressing cells versus those expressing the scrambled siRNA. Scale
bar=15 .mu.m.
[0044] FIG. 21. The small molecule inhibitor for FAK, TAE226,
reduces FAK activation and stretch-induced MMP-10 and MMP-12
expression in cultured podocytes. A) Podocytes were cultured on
placental laminin in the presence or absence of TAE226 overnight.
Extracts were prepared and analyzed by western blot for expression
of pFAK397 and total FAK. FAK activation was also analyzed by
western blot of podocyte extracts from stretched and non-stretched
cells, demonstrating that biomechanical stretching directly
activates FAK. B-C) Cells were treated or not with TAE226 under
static and stretched conditions and mRNA analyzed by real time
qRT-PCR for the indicated transcripts.
[0045] FIG. 22. Biomechanical stretching activates NF-kappaB which
regulates MMP-10 expression in cultured podocytes. A) NF-kappaB
localizes primarily to the cytosol in non-stretched cultured
podocytes. B) Subjecting the cells to cyclic biomechanical
stretching results in the nuclear localization of NF-kappaB, which
is consistent with its activation. C) Stretch-mediated induction of
MMP-10 is blocked by addition of a peptide inhibitor for NF-kappaB
to the culture medium. Scale bar=20 .mu.m.
[0046] FIG. 23. Treatment of Alport mice with the small molecule
inhibitor for FAK, TAE226, blocks FAK activation, significantly
reduces glomerular expression of MMP-9, -10, and -12, and
ameliorates proteinuria and blood urea nitrogen levels. 129 Sv/J
autosomal Alport mice were treated with TAE226 from 2 to 7 weeks of
age. A-F) While pFAK397 immunostaining is present in podocytes
adjacent to laminin 211-immunopositive basement membranes in
vehicle treated mice, it is absent in mice treated with TAE226,
indicating effective blockade. G) Real time qRT-PCR analysis of
glomerular RNA shows significant reduction in expression of MMP-9,
MMP-10, and MMP-12 in TAE226 treated mice relative to those given
vehicle. G-I) Significant amelioration of proteinuria and BUN in
treated mice at 6 weeks of age, indicative of improved glomerular
function, however the values loose significance at 7 week of age.
Scale bar=15 .mu.m.
[0047] FIG. 24. Treatment of Alport mice with TAE226 reduces
mesangial process invasion of the glomerular capillary loops,
ameliorates GBM ultrastructural dysmorphology, and significantly
reduces pFAK activation and migratory potential of primary cultured
mesangial cells. A-F) The same mice as in FIG. 23 were dual
immunostained with the GBM marker laminin .alpha.5 (A and D), and
the mesangial cell marker integrin .alpha.8 (B and E). (C) and (F)
represent a merging of results obtained from staining with laminin
.alpha.5 and integrin .alpha.8. Arrowheads in C denote regions
where invasion of the capillary loops by mesangial processes is
evident (inserts in B and C). This characteristic is markedly
reduced in the TAE226-treated glomeruli where integrin .alpha.8
immunostaining is restricted to the mesangial angles (inserts in E
and F). G-J) Transmission electron microscopic analysis (G-I) shows
that TAE226 treatment (I) reduces the ultrastructural damage to the
GBM normally present by 7 weeks of age in this model (H).
Amelioration of GBM dysmorphology is generally observed (J).
Treatment of primary cultured mesangial cells with TAE226
significantly reduces their migratory potential relative to
untreated cells (I). K) Dose response for FAK inhibition by TAE226
in cultured mesangial cells. L) Northern blot for pFAk and
.beta.actin. F, scale bar=15 m; J, scale bar=2 .mu.m.
[0048] FIG. 25. Treatment of Alport mice with TAE226 ameliorates
interstitial fibrosis and monocyte infiltration. Kidney
cryosections from wild type and Alport mice that were either
treated with vehicle or TAE226 were immunostained with antibodies
specific for fibronectin (A-C) or the monocyte marker, CD11b (D-F).
The accumulation of fibronectin in the interstitium, indicative of
fibrosis, while abundant in Alport mice (B) is not apparent in
Alport mice treated with TAE226 (C), which appear similar to wild
type mice (A). Similarly, monocyte infiltration, as indicated by
CD11b immunopositive cells, is readily apparent in Alport mice (E).
In TAE226-treated Alport mice (F), however, the abundance of
monocytes is similar to that in wild type mice (D), which are
resident cells rather than infiltrating cells. Scale bar=50
.mu.m.
[0049] FIG. 26. Induction of hypertension and hypotension in Alport
mice. C57Bl/6 X-linked Alport mice were made hypertensive by giving
them the L-NAME salts in their drinking water from 4 to 7 weeks of
age. Normotensive mice were given plain drinking water. Blood
pressures were measured longitudinally using the CODA-2 tail cuff
system.
[0050] FIG. 27. Hypertension induces endothelin-1 in Alport
glomerular endothelial cells. Cryosections were dual immunostained
with antibodies specific for either endothelin-1 (ET-1) (A, D, G,
and J) or CD31 (a marker for endothelial cells) (B, E, and H).
Panels C, I, and L are a merging of results from staining with
endothelin-1 and CD31. Elevated expression of endothelin-1 is
clearly evident in glomeruli from hypertensive mice relative to
normotensive mice (compare G and J). This was not observed in wild
type mice (compare A and D). Co-localization of endothelin-1 with
CD31 demonstrates this induction is coming from the endothelial
cell compartment.
[0051] FIG. 28. ET-1 protein expression is elevated in glomeruli
from Alport mice relative to age/strain-matched wild type mice.
Glomeruli were isolated from 7 week old 129 Sv autosomal Alport
mice and wild type mice. Lysates were analyzed by western blots and
probed with anti-ET-1 antibodies.
[0052] FIG. 29. Endothelin A receptor is the primary endothelin
receptor on mouse mesangial cells. A) Kidney cryosections from wild
type mice were dual stained with antibodies specific for the
endothelin A receptor and the mesangial cell marker integrin
.alpha.8. Staining in a single glomerulus is shown. The merged
image shows that the endothelin A receptor staining is primarily in
the mesangial cell compartment. The lower panels show that
Endothelin B receptors are principally expressed on podocytes,
consistent with earlier reports (Wendel et al., 2006).
Alpha-actinin-4 is used as a podocyte marker. B) Western blots from
cultured mesangial cells and podocytes confirm that ETAR is
robustly expressed on mesangial cells, while ETBR is not detected.
Cultured podocytes and glomerular outgrowths (cultured for 24 hours
after glomerular isolation) express both ETAR and ETBR.
[0053] FIG. 30. Treatment of mesangial cells with endothelin-1
activates CDC42 and induces the formation of drebrin-positive actin
microspikes; microspikes and CDC42 activation are inhibited by
pre-treatment of cells with Sitaxentan. A) Cultured mesangial cells
were serum starved, pretreated for 1 hour with or without
Sitaxentan, treated for 30 minutes with endothelin-1, fixed with
acetone, and dual stained with anti-drebrin antibodies (red) and
phalloidin (green). Drebrin-positive microspikes (filopodia,
denoted by arrowheads) are highly abundant on the
endothelin-treated cells, but not detected when the cells are
pre-treated with Sitaxentan. B) Cells were treated as in A, then
lysates prepared and assayed by ELISA for GTP-CDC42. Endothelin
treatment significantly activates CDC42 in the cultured mesangial
cells, and its activation is inhibited by pre-treatment of cell
with Sitaxentan.
[0054] FIG. 31. Endothelin A receptor blockade prevents mesangial
process invasion of glomerular capillaries and ameliorates GBM
damage. 129 Alport animals were treated with the endothelin A
receptor specific blocking agent Sitaxsentan from 2 weeks to 7
weeks of age. A) Dual staining demonstrates absence of integrin
.alpha.8 immunostaining in the glomerular capillaries, which are
dual stained with either anti-laminin .alpha.2 or anti-laminin
.alpha.5 antibodies. Arrows denote integrin .alpha.8
immunopositivity in the capillary loops of the glomeruli from
untreated Alport mice, and the relative absence of integrin
.alpha.8 immunopositivity in the sitaxentan-treated mice. B)
Sitaxsentan ameliorates GBM dysmorphology, largely normalizing the
irregular thickening and thinning observed for the GBM of 7 week
old Alport mice.
[0055] FIG. 32. Quantitative analysis of integrin .alpha.8
immunolabeling in the GBM demonstrates extensive filopodial
invasion in 7 week Alport glomeruli that is prevented by treatment
of animals with Sitaxentan. At least 6 glomeruli from at least 3
independent animals per group were analyzed by quantifying red
fluorescence in circumferential capillary loops (defined by laminin
.alpha.5 immunostaining in green) using NIH image J software.
Mesangial angles were excluded. A) An example of how the
capillaries were partitioned for these measures. B) Immunogold
labeling for integrin .alpha.8 in a filopodial cross-section on the
sub-endothelial aspect of an Alport capillary loop (arrow). Note
the absence of immunogold labeling in the podocyte pedicles
(asterisks) and the fenestrated endothelium (arrowheads). C)
Quantitative results for red fluorescence in the capillary loops.
The quantitative analysis clearly demonstrates extensive integrin
.alpha.8 immunolabeling in the glomerular capillaries of Alport
mice, which is significantly reduced (to near wild type control
levels) in Alport mice treated with Sitaxentan.
[0056] FIG. 33. Sitaxentan treatment of Alport mice significantly
delays the onset and progression of proteinuria. Urine was
collected at the indicated time points and analyzed for albumin
using an ELISA kit. Albumin measures were normalized to urinary
creatinine. Note that measurable albumin in the Sitaxentan-treated
mice was not detected until 6 weeks of age indicating a delayed
onset of glomerular disease.
[0057] FIG. 34. Sitaxentan treatment ameliorates interstitial
fibrosis and monocytic infiltration in Alport kidneys. Cryosections
from Sitaxentan-treated and vehicle treated wild type and Alport
mice were immunostained with antibodies specific for fibronectin (a
marker for fibrosis) (A, D, and G) and CD11b (a marker for
interstitial monocytes) (B, E, and H). Panels C, F, and I are a
merging of results from staining with FN and CD11b. Note the
complete absence of fibrosis and interstitial monocytes in the
treated mice.
[0058] FIG. 35. Bosentan treatment reduces nRNA expression of
MMP-9, MMP-10, MMP-12, MCP-1, and TGF.beta.1 in glomeruli from
Alport mice. Glomerular RNA from Bosentan-treated and
vehicle-treated mice was analysed by real time RT-PCR for MMP-9 and
MMP-12 transcripts (A), MMP-10 transcripts (B), and MCP-1 and
TGF.beta.1 transcripts (C). MMP (matrix metalloproteinase), MCP-1
(monocyte chemoattractant protein-1), TGF.beta.1 (transforming
growth factor beta-1).
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0059] Alport syndrome (also referred to as hereditary nephritis)
is a genetic disorder characterized by abnormalities in the
basement membranes of the glomerulus (leading to hematuria,
glomerulosclerosis, and end-stage kidney disease (ESRD)), cochlea
(causing hearing loss), and eye (resulting in lenticonus and
perimacular flecks). Alport syndrome is a primary basement membrane
disorder caused by mutations in the collagen type IV COL4A3,
COL4A4, or COL4A5 genes. Mutations in any of these genes prevent
the proper production or assembly of the type IV collagen network,
which is an important structural component of basement membranes in
the kidney, inner ear, and eye. Basement membranes are thin,
sheet-like structures that separate and support cells in many
tissues. The abnormalities of type IV collagen in kidney basement
membranes leads to irregular thickening and thinning and splitting
of basement membranes, causing gradual scarring of the kidneys.
Alport Syndrome causes progressive kidney damage. The glomeruli and
other normal kidney structures such as tubules are gradually
replaced by scar tissue, leading to kidney failure. Deafness and an
abnormality in the shape of the lens called anterior lenticonus are
other important features of Alport Syndrome. People with anterior
lenticonus may have problems with their vision and may develop
cataracts. The prevalence of Alport syndrome is estimated at
approximately 1 in 5,000 births and it is estimated that the
syndrome accounts for approximately 2.1 percent of pediatric
patients with ESRD. Currently there is no specific treatment for
Alport Syndrome; treatments are symptomatic. Patients are advised
on how to manage the complications of kidney failure and the
proteinuria that develops is often treated with ACE inhibitors.
Once kidney failure has developed, patients are given dialysis or
can benefit from a kidney transplant, although this can cause
problems. The body may reject the new kidney as it contains normal
type IV collagen, which may be recognized as foreign by the immune
system. Thus there is a need for improved therapeutic agents for
the treatment of individuals with Alport syndrome, especially for
the treatment of presymptomatic individuals, before the onset of
proteinuria.
[0060] Alport syndrome is characterized by delayed onset
progressive glomerulonephritis associated with sensorineural
hearing loss and retinal flecks (Kashtan and Michael, 1996, Kidney
Int; 50(5):1445-1463). The most common form (80%) is X-linked and
caused by mutations in the type IV collagen COL4A5 gene (Barker et
al., 1990, Science; 8; 248(4960):1224-7). The two autosomal forms
of the disease account for the remaining 20% of Alport patients,
and result from mutations in the COL4A3 and COL4A4 genes (Mochizuki
et al., 1994, Nat Genet; 8(1):77-81). The .alpha.3(IV),
.alpha.4(IV) and .alpha.5(IV) proteins form a heterotrimer and is
assembled into a subepithelial network in the glomerular basement
membrane that is physically and biochemically distinct from a
subendothelial type IV collagen network comprised of .alpha.1(IV)
and .alpha.2(IV) heterotrimers (Kleppel et al., 1992, J Biol Chem;
267(6):4137-4142). Mutations in any one of the three type IV
collagen genes that cause Alport syndrome results in the absence of
all three proteins in the GBM due to an obligatory association to
form functional heterotrimers (Kalluri and Cosgrove, 2000, J Biol
Chem; 275(17):12719-12724). Thus, the net result for all genetic
forms of Alport syndrome is the absence of the .alpha.3(IV)
.alpha.4(IV) .alpha.5(IV) subepithelial collagen network, resulting
in a GBM type IV collagen network comprised only of .alpha.1(IV)
and .alpha.2(IV) heterotrimers.
[0061] This change in basement membrane composition does not result
in immediate pathology. The GBM appears to function adequately for
the first few years of life and sometimes past the first decade
(Kashtan et al., 1998, Pediatr Nephrol; 12(4):269-27). This delayed
onset predicts a triggering mechanism for glomerular disease
initiation and a theoretical window for therapeutic intervention
that may arrest or significantly ameliorate Alport renal disease in
its earliest stages.
[0062] Alport syndrome may result from mutations in type IV
collagen COL4A3, COL4A4, or COL4A5 genes. These mutations may be
either autosomal recessive (mutations in either COL4A3 or COL4A4
genes (Mochizuki et al., 1994, Nat Genet; 8(1):77-81)) or X-linked
(mutations in COL4A5 (Barker et al., 1990, Science;
248(4960):1224-7)). Mutations in any of these genes results in the
absence of all three collagens (.alpha.3(IV), .alpha.4(IV), and
.alpha.5(IV) in the GBM type IV collagen network due to an
obligatory association to form heterotrimers. The result is a
thinner and less cross-linked GBM collagen network resulting in
delayed onset progressive glomerulonephritis. Until the
observations of the present invention, the molecular trigger for
disease onset was unknown.
[0063] Alport syndrome is also known as congenital hereditary
hematuria, hematuria-nephropathy-deafness syndrome, hematuric
hereditary nephritis, hemorrhagic familial nephritis, hemorrhagic
hereditary nephritis, hereditary familial congenital hemorrhagic
nephritis, hereditary hematuria syndrome, hereditary interstitial
pyelonephritis, and hereditary nephritis.
[0064] With the present invention, it has been discovered that the
pathology of Alport glomerular disease is based on an entirely
different mechanism. Alport syndrome results from a change in the
type IV collagen composition in the glomerular basement membrane
where the normally present .alpha.3(IV)/.alpha.4(IV)/.alpha.5(IV)
network is absent, and thus the type IV collagen composition of the
GBM is thus comprised of a network of .alpha.1(IV) and .alpha.2(IV)
chains. This latter network is known to contain fewer interchain
crosslinks and is thinner than a normal GBM, and thus we expect
that the GBM would have a greater elasticity under normal
glomerular pressures, which are very high relative to blood
pressures in most tissues in the body. This enhanced elasticity
imparts unusually high biomechanical stresses on the cells that
adhere to the GBM, including podocytes, endothelial cells, and
mesangial cells. Consistent with this, Alport mice made
hypertensive by including salt in the drinking water showed higher
proteinuria, elevated levels of glomerular matrix metalloproteinase
expression, and accelerated damage to the GBM (Meehan et al., 2009,
Kidney Int; 76(9):968-76).
[0065] The present invention shows that hypertensive mice express
much higher levels of endothelin-1, specifically in the endothelial
cells, and endothelin A receptors, specifically on mesangial cells.
Activation of endothelin receptors on glomerular mesangial cells,
specifically endothelin receptor A, has been linked to activation
of the rho-GTPases, Rac1 and CDC42 (reviewed in Sorokin, 2011,
Contrib Nephrol; 172:50-62). Classically, CDC42 activation results
in filopodial formation in cultured cells. The present examples
show crosstalk between Rac1 and CDC42 in cultured mesangial cells
and demonstrated mesangial filopodial invasion of the glomerular
capillary tufts, and showed that blocking RAC1 ameliorated this
invasion, restored GBM ultrastructural pathology, and reduced
pathologic gene expression in the glomeruli from Alport mice (see
Example 1 and Zallocchi et al., 2013, Am J Pathol;
183(4):1269-802013). These invading mesangial filopodia secrete
laminin 211, which activates focal adhesion kinase (FAK) on
glomerular podocytes. Blocking FAK reduces pathologic expression of
MMPs and ameliorates GBM damage. The present examples also show
that endothelin blockade also reduces pathologic expression of MMPs
as well.
[0066] Collectively, the present invention defines a mechanism
whereby biomechanical strain induces expression of endothelin-1 in
glomerular endothelial cells and endothelin A receptor on mesangial
cells in Alport glomeruli. Endothelin A activation leads to
mesangial filopodial invasion of the glomerular capillary tufts.
The filopodia deposit laminin 211, which activates FAK in
podocytes, resulting in marked elevation of pro-pathologic genes
including MMP-10 and MMP-12. These MMPs proteolytic degrade the GBM
driving glomerulosclerosis. Blocking endothelin receptor activation
using Bosentan or activation of cytoskeletal dynamics using Rac1
inhibitors arrests the invasion of the capillary tufts by mesangial
filopodia. This activation of mesangial filopodia invasion has
never been described before and is thus a new etiology.
[0067] The present invention provides for the administration of an
endothelin receptor antagonist to prevent the damage induced by
biomechanical strain and to prevent the initiation of disease
pathology. The present invention provides new methods of use for
endothelin receptor antagonists. Blocking the activation of this
process with an endothelin receptor antagonist such as bosentan
represents a new use for such drugs.
[0068] The present invention includes methods of treating Alport
syndrome in a subject by the administration of a RAC1 inhibitor
and/or a CDC42 inhibitor. The administration of a RAC1 inhibitor
and/or a CDC42 inhibitor may result in, for example, inhibiting
migration of mesangial cells, inhibiting irregular deposition of
mesangial laminin 211 in the GBM, inhibiting invasion of the
capillary loops by mesangial cell processes, inhibiting mesangial
filopodial invasion of the glomerular capillary tuft, and/or
preventing, or slowing the onset of proteinuria.
[0069] The present invention includes methods of preventing,
slowing, and/or managing glomerular disease progression in a
subject diagnosed with Alport syndrome by the administration of a
RAC1 inhibitor and/or a CDC42 inhibitor.
[0070] The present invention includes methods of treating
glomerulonephritis associated with Alport syndrome in a subject by
administering a RAC1 inhibitor and/or a CDC42 inhibitor.
[0071] The present invention includes methods of treating kidney
injury due to biomechanical strain in Alport syndrome by
administering a RAC1 inhibitor and/or a CDC42 inhibitor.
[0072] The present invention includes methods of inhibiting
deposition of laminin 211 in the glomerular basement membrane (GBM)
by administering a RAC1 inhibitor and/or a CDC42 inhibitor. The
laminins are major proteins in the basal lamina, a layer of the
basement membrane, a protein network foundation for most cells and
organs. Laminins are heterotrimeric proteins that contain an
.alpha.-chain, a .beta.-chain, and a .gamma.-chain, found in five,
four, and three genetic variants, respectively. The laminin
molecules are named according to their chain composition. Thus,
laminin-511 contains .alpha.5, .beta.1, and .gamma.1 chains
(Aumailley et al., 2005, Matrix Biol; 24(5):326-32). Fourteen other
chain combinations have been identified in vivo. Laminin-211
(composed of .alpha.2, .beta.1 and .gamma.1 chains (Ehrig et al.,
1991, PNAS; 87:3264-3268) is the main laminin isoform in skeletal
muscle (Leivo and Engvall, 1988, PNAS; 85:1544-1588; and Patton,
1997, J Cell Biol; 139:1507-1521) and identification of laminin
.alpha.2 chain mutations in a severe form of congenital muscular
dystrophy (merosin-deficient congenital muscular dystrophy; MDC1A)
established the importance of laminin-211 for normal muscle
function (Helbling-Leclerc et al., 1995, Nat Genet; 11:216-218).
The present invention demonstrates for the first time, the role of
the deposition of laminin 211 in the glomerular basement membrane
(GBM) in the pathogenesis of Alport syndrome. Its role is to
activate focal adhesion kinase in glomerular podocytes. As shown in
the examples included herewith, laminin 211 mediates FAK activation
in Alport podocytes and FAK inhibitors ameliorate Alport kidney
disease. This demonstrates that laminin 211 in the GBM activates
FAK on podocytes which results in pro-pathologic changes in gene
expression.
[0073] The present invention includes methods of inhibiting
mesangial cell process invasion of the glomerular capillary loop of
the kidney by administering a RAC1 inhibitor and/or a CDC42
inhibitor. RAC1 (also referred to herein as Rac1) is a member of
the Rac subfamily (Rac1-Rac4) of the Rho family of GTPases. Members
of this superfamily appear to regulate a diverse array of cellular
events, including the control of cell growth, cytoskeletal
reorganization, and the activation of protein kinases. Together
with Rho (regulator of stress fibers) and Cdc42 (regulator of
filopodia), Rac modulates the formation of focal adhesion (FA)
complexes; membrane ruffles and lamellipodia that contribute to
important cell functions related to cell attachment and
movement.
[0074] The methods of the present invention may be used for the
presymptomatic treatment of individuals, with the administration of
a RAC1 inhibitor and/or a CDC42 inhibitor beginning after the
determination or diagnosis of Alport syndrome, prior to the onset
of symptoms, such as for, example, proteinuria. The diagnosis of
Alport syndrome in an individual may be made, for example, by
family medical history, genetic testing, immunodiagnostic skin
biopsy testing, and/or molecular diagnostic marker testing. Methods
of the present invention may also include one or more steps of
obtaining a diagnosis of Alport syndrome by the use of one or more
such diagnostic means.
[0075] A RAC1 inhibitor or a CDC42 inhibitor may block the
activation of RAC1/CDC42 members of the rho family of small
GTPases. Any of a wide variety of RAC1 inhibitors or CDC42
inhibitors may be used with the methods of the present invention.
In some aspects, a RAC1 inhibitor or a CDC42 inhibitor may include
a small molecule inhibitor. In some aspects, a RAC1 inhibitor or a
CDC42 inhibitor may include a biologic, such as, for example, an
antibody or receptor polypeptide.
[0076] In some aspects, a RAC1 inhibitor or a CDC42 inhibitor is an
antibody that binds to endothelin-1 and/or the endothelin A
receptor. In some aspects, such an antibody inhibits, blocks,
and/or neutralizes one or more functions of endothelin-1 and/or the
endothelin A receptor. Antibodies that bind to endothelin-1 or the
endothelin A receptor can be produced and characterized by any of a
variety of means known to the skilled artisan. Likewise, antibodies
that inhibit and/or neutralize one of more functions of
endothelin-1 or the endothelin A receptor can also be produced and
characterized by any of a variety of means known to the skilled
artisan.
[0077] As will be understood by those in the art, the term
"antibody" extend to all antibodies from all species, and antigen
binding fragments thereof, including dimeric, trimeric and
multimeric antibodies; bispecific antibodies; chimeric antibodies;
human and humanized antibodies; recombinant and engineered
antibodies, and fragments thereof. The term "antibody" is thus used
to refer to any antibody-like molecule that has an antigen binding
region, and this term includes antibody fragments such as, for
example, Fab', Fab, F(ab').sub.2, single domain antibodies (DABs),
Fv, scFv (single chain Fv), linear antibodies, diabodies, and the
like. The techniques for preparing and using various antibody-based
constructs and fragments are well known in the art.
[0078] In certain embodiments, the antibodies employed may be
"humanized" antibodies. Humanized" antibodies are generally
chimeric monoclonal antibodies from mouse, rat, or other non-human
species, bearing human constant and/or variable region domains.
Various humanized monoclonal antibodies for use in the present
invention will be chimeric antibodies wherein at least a first
antigen binding region, or complementarity determining region
(CDR), of a mouse, rat or other non-human monoclonal antibody is
operatively attached to, or "grafted" onto, a human antibody
constant region or "framework." Humanized monoclonal antibodies for
use herein may also be monoclonal antibodies from non-human species
wherein one or more selected amino acids have been exchanged for
amino acids more commonly observed in human antibodies. This can be
readily achieved through the use of routine recombinant technology,
particularly site-specific mutagenesis.
[0079] Entirely human antibodies may also be prepared and used in
the present invention. Such human antibodies may be obtained from
healthy subjects by simply obtaining a population of mixed
peripheral blood lymphocytes from a human subject, including
antigen-presenting and antibody-producing cells, and stimulating
the cell population in vitro.
[0080] In some aspects, a RAC1 inhibitor or a CDC42 inhibitor may
be a small molecule inhibitor. For example, a RAC1 inhibitor or a
CDC42 inhibitor may include, but is not limited to, NSC23766 and
derivatives thereof (Gao et al., 2004, PNAS; 101:7618-7623), EHT
1864 and derivatives thereof (Shutes et al., 2007, J Biol Chem;
282:35666-35678), W56 (Gao et al., 2001, J Biol Chem; 276:47530),
F56 (Gao et al., 2001, J Biol Chem; 276:47530), and any of the RAC1
inhibitors described by Ferri et al. (J Med Chem 2009;
52(14):4087-90) and Hernandez et al. (P R Health Sci J 2010;
29(4):348-356). In some aspects of the methods described herein, a
RAC1 inhibitor may be NSC23766 or a derivative thereof. Human CDC42
is a small GTPase of the Rho-subfamily, which regulates signaling
pathways that control diverse cellular functions including cell
morphology, migration, endocytosis and cell cycle progression.
[0081] Any of a wide variety of CDC42 inhibitors may be used with
the methods described herein, including, but not limited to,
secramine (Pelish et al., 2006, Nat Chem Biol; 2(1):39-46), ML141
(Surviladze et al., "A Potent and Selective Inhibitor of Cdc42
GTPase," Probe Reports from the NIH Molecular Libraries Program
[Internet], Bethesda (Md.): National Center for Biotechnology
Information (US); 2010), or an endothelin receptor antagonist, such
as, for example, bosentan, ambrisentan, or derivatives thereof.
[0082] In some aspects of the methods described herein, a RAC1
inhibitor or a CDC42 inhibitor may include an endothelin receptor
antagonist. Such an endothelin receptor antagonist includes, but is
not limited to, small molecule antagonists and biologics, such as
for example, an antibody or receptor polypeptide.
[0083] Endothelin receptor antagonists include, for example,
bosentan, a dual endothelin receptor antagonist, is currently
indicated mainly for the treatment of pulmonary arterial
hypertension (PAH) (see Rubin et al., 2002, N Engl J Med; 346(12):
896-903). In 2007, bosentan was also approved in the European Union
for reducing the number of new digital ulcers in patients with
systemic sclerosis and ongoing digital ulcer disease. It is also
known by the trade name TRACLEER.RTM. (Actelion Pharmaceuticals US,
Inc.), is designated chemically as
4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)[2,2]-bipyrimid-
in-4-yl]-benzenesulfonamide monohydrate, has the chemical formula
C.sub.27H.sub.31N.sub.5O.sub.7S, and the CAS Registry number
157212-55-0.
[0084] While bosentan has been used experimentally to treat
diabetic nephropathy (Ritz and Wenzel, 2010, J Am Soc Nephrol;
21(3):392-4), the molecular basis for this use is that endothelin
causes vasoconstriction through its activation of endothelin
receptors, and thus blockade of these receptors results in
vasodilation and a drop in blood pressure in the glomerulus, thus
reducing proteinuria. Since these blocking agents are
mechanistically distinct from angiotensin converting enzyme (ACE)
inhibitors, it has been thought that a combination therapy could be
quite beneficial; similar to the FDA approved use of Bosentan to
treat pulmonary hypertension, which is also often employed in
combination with ACE inhibitors.
[0085] Endothelin receptor antagonists include, for example,
ambrisentan, an endothelin receptor antagonist selective for the
type A endothelin receptor (ETA) (reviewed by Vatter and Seifert,
2006, Cardiovasc Drug Rev; 24(1):63-76), is currently indicated for
the treatment of pulmonary arterial hypertension (PAH) (see
Frampton, 2011, Am J Cardiovascul Drugs; 11(4):215-226). In the
United States it is also known by the trade name LETARIS.RTM., is
also known as Volobris, and pulmonest, is designated chemically as
(2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropanoic
acid, CAS 7036-94-1, and has the CAS Registry number
177036-94-1.
[0086] Endothelin receptor antagonists include, for example,
macitentan (trade name OPSUMIT.RTM.; designated chemically as
N-[5-(4-Bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-pyrimidin-
yl]-N'propylsulfamide), an orally available endothelin receptor
antagonist (ERA) indicated for the treatment of pulmonary arterial
hypertension.
[0087] Endothelin receptor antagonists include, for example,
atrasentan (chemically designated as
2R,3R,4S)-4-(1,3-benzodioxol-5-yl)-1-[2-(dibutylamino)-2-oxoethyl]-2-(4-m-
ethoxyphenyl)pyrrolidine-3-carboxylic acid), an endothelin receptor
antagonist selective for subtype A (ETA). While other drugs of this
type (sitaxentan, ambrisentan) exploit the vasoconstrictive
properties of endothelin and are mainly used for the treatment of
pulmonary arterial hypertension, atrasentan blocks endothelin
induced cell proliferation.
[0088] Endothelin receptor antagonists include, for example,
sitaxentan (also known as TBC-11251 sodium salt, Thelin; chemically
designated as
N-(4-chloro-3-methyl-1,2-oxazol-5-yl)-2-[2-(6-methyl-2H-1,3-benzodioxol-5-
-yl)acetyl]thiophene-3-sulfonamide), a small molecule that blocks
the action of endothelin (ET) on the endothelin-A (ETA) receptor
selectively (by a factor of 6000 compared to the ETB). It is a
sulfonamide class endothelin receptor antagonist (ERA).
[0089] Endothelin receptor antagonists include, for example,
darusentan, an endothelin-1 receptor A antagonist, chemically
designated as
(2S)-2-(4,6-Dimethoxypyrimidin-2-yl)oxy-3-methoxy-3,3-di(phenyl)propanoic
acid).
[0090] In some aspects of the methods described herein, an
endothelin receptor antagonist blocks CDC42 activation in
glomerular mesangial cells. This is well established in cultured
cells (Chadi and Sorokin, 2006, Exp Biol Med; 6:761). Three main
kinds of ERAs are known: selective ET.sub.A receptor antagonists
(sitaxentan, ambrisentan (LETAIRIS), atrasentan, BQ-123,
zibotentan), which affect endothelin A receptors; dual antagonists
(bosentan (TRACLEER), macitentan, tezosentan), which affect both
endothelin A and B receptors; and selective ET.sub.B receptor
antagonists (BQ-788 and A192621).
[0091] In some aspects of the methods described herein, an
endothelin receptor antagonist is bosentan or a derivative
thereof.
[0092] In some aspects of the methods described herein, an
endothelin receptor antagonist is ambrisentan or a derivative
thereof.
[0093] In some aspects of the methods described herein, an
endothelin receptor antagonist is macitentan or a derivative
thereof.
[0094] In some aspects of the methods described herein, an
endothelin receptor antagonist is altrasentan or a derivative
thereof.
[0095] With the present invention, a novel mechanism responsible
for the pathology of Alport glomerular disease has been discovered.
This mechanism, described in more detail in the examples included
herewith, includes one or more of the following:
[0096] changes in basement membrane type IV collagen composition
result in distension of the capillary in response to normal blood
pressure;
[0097] biomechanical strain results in activation of endothelin-1
expression in glomerular endothelial cells;
[0098] binding of endothelin-1 to endothelin receptors on mesangial
cells activates the rho GTPases Rac1 and CDC42, resulting in the
activation of actin cytoskeletal dynamics and the invasion of the
glomerular capillaries by mesangial filopodia;
[0099] mesangial filopodia secrete mesangial matrix molecules into
the GBM microenvironment, including laminin 211;
[0100] laminin 211 directly activates focal adhesion kinase on
glomerular podocytes;
[0101] FAK activation results in NFkappaB-mediated induction of
MMPs and pro-inflammatory cytokines that degrade the GBM,
progressively resulting in classical Alport ultrastructural
abnormalities (irregular thickening and thinning with
multi-lamination) and proteinuria; and/or
[0102] progressive glomerular disease results in interstitial
fibrosis.
With this newly gained understanding of the mechanism responsible
for the pathology of Alport glomerular disease, agents that block
one or more of the mechanisms listed above may be used to prevent
and/or treat the symptoms of Alport disease.
[0103] The present invention also includes in vitro and in vivo
assays for the screening and identification of agents with
endothelin receptor antagonist activity for use in the treatment of
Alport syndrome. Such assays include, but are not limited to, any
one of the various cell culture and animal model systems described
herein. In vitro assays include, for example, cultured primary
mesangial cells (for example, as described by Cosgrove et al.,
2008, Am J Pathol; 172: 761-773), cultured podocytes, and
conditional immortalized glomerular epithelial cells (GEC's) (Rao
et al., 2006, Am J Pathol; 169: 32-46). The treatment (contacting)
of such cultured cells with endothelin-1 induces the formation of
drebrin-positive filopodial microspikes. Potential endothelin
receptor antagonist activity of an agent may be identified and/or
assayed by pretreatment (contacting) of the cells with the agent,
with a potential endothelin receptor antagonist inhibiting,
reducing and/or blocking the formation of microspikes in comparison
to cells not pretreated with the agent. Any such assay may also
include appropriate controls, including, but not limited to
negative and/or positive controls.
[0104] With the method of the present invention, one or more
additional therapeutic modalities may be administered along with
one or more agents of the present disclosure. In some aspects of
the present invention, the administration of agents of the present
disclosure may allow for the effectiveness of a lower dosage of
other therapeutic modalities when compared to the administration of
the other therapeutic modalities alone, providing relief from the
toxicity observed with the administration of higher doses of the
other modalities. One or more additional therapeutic agents may be
administered before, after, and/or coincident to the administration
of agents of the present disclosure. Agents of the present
disclosure and additional therapeutic agents may be administered
separately or as part of a mixture of cocktail. As used herein, an
additional therapeutic agent may include, for example, an agent
whose use for the treatment of Alport syndrome, kidney disease,
kidney failure, and/or proteinuria is known to the skilled artisan.
For example, an angiotensin-converting enzyme (ACE) inhibitor, such
as ramipril or analapril, may be administered.
[0105] As used herein "treating" or "treatment" can include
therapeutic and/or prophylactic treatments. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, decreasing the rate of
disease progression, amelioration or palliation of the disease
state, and remission or improved prognosis.
[0106] The agents of the present disclosure can be administered by
any suitable means including, but not limited to, for example,
oral, rectal, nasal, topical (including transdermal, aerosol,
buccal and sublingual), vaginal, parenteral (including
subcutaneous, intramuscular, intravenous and intradermal),
intravesical, or injection. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous, intraperitoneal, and intratumoral
administration. In this connection, sterile aqueous media that can
be employed will be known to those of skill in the art. Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, and general
safety and purity standards as required by the FDA. Such
preparation may be pyrogen-free.
[0107] For enteral administration, the inhibitor may be
administered in a tablet or capsule, which may be enteric coated,
or in a formulation for controlled or sustained release. Many
suitable formulations are known, including polymeric or protein
microparticles encapsulating drug to be released, ointments, gels,
or solutions which can be used topically or locally to administer
drug, and even patches, which provide controlled release over a
prolonged period of time. These can also take the form of
implants.
[0108] The present invention includes compositions of one or more
of the inhibitors described herein. A composition may also include,
for example, buffering agents to help to maintain the pH in an
acceptable range or preservatives to retard microbial growth. Such
compositions may also include a pharmaceutically acceptable
carrier. As used herein, the term "pharmaceutically acceptable
carrier" refers to one or more compatible solid or liquid filler,
diluents or encapsulating substances which are suitable for
administration to a human or other vertebrate animal. The
compositions of the present disclosure are formulated in
pharmaceutical preparations in a variety of forms adapted to the
chosen route of administration.
[0109] Therapeutically effective concentrations and amounts may be
determined for each application herein empirically by testing the
compounds in known in vitro and in vivo systems, such as those
described herein, dosages for humans or other animals may then be
extrapolated therefrom. With the methods of the present disclosure,
the efficacy of the administration of one or more agents may be
assessed by any of a variety of parameters known in the art.
[0110] It is understood that the precise dosage and duration of
treatment is a function of the disease being treated and may be
determined empirically using known testing protocols or by
extrapolation from in vivo or in vitro test data. It is to be noted
that concentrations and dosage values may also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
compositions and methods.
[0111] An agent of the present disclosure may be administered at
once, or may be divided into a number of smaller doses to be
administered at intervals of time. For example, an agent of the
present disclosure may be administered twice a day, three times a
day, four times a day, or more. For example an agent of the present
disclosure may be administered every other day, every third day,
once a week, every two weeks, or once a month. In some
applications, an agent of the present disclosure may be
administered continuously, for example by a controlled release
formulation or a pump.
[0112] It is understood that the precise dosage and duration of
treatment is a function of the disease being treated and may be
determined empirically using known testing protocols or by
extrapolation from in vivo or in vitro test data. It is to be noted
that concentrations and dosage values may also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
compositions and methods.
[0113] In some applications, administration on agent of the present
disclosure may be short term or long term. In some aspects, long
term administration may be for weeks, months, years, or
decades.
[0114] In some applications, administration on agent of the present
disclosure may be at a dosage similar to the accepted dosage for
previously known applications. For example, an agent such as
bosentan or a derivative thereof may be administered at a dosage
similar to the dosage that is administered for the treatment of
pulmonary arterial hypertension (PAH) at about 62.5, about 125, or
about 250 mg/day. For example, an agent such as ambrisentan or a
derivative thereof may be administered at a dosage similar to the
dosage that is administered for the treatment of pulmonary arterial
hypertension (PAH) at about 2.5 to about 10 mg/day.
[0115] In some applications, administration on agent of the present
disclosure may be at a dosage considerably less than the accepted
dosage for previously known applications. For example, dosage may
be 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/250, 1/500, 1/1,000,
1/2,500, 1/5,000, 1/10,000, 1/25,000, 1/50,000, or 1/100,000 the
accepted dosage.
[0116] For example, an agent, such as, for example, bosentan or
ambrisentan, or a derivative thereof, may be administered at a
dosage of about 0.1, about 0.2, about 0.25, about 0.4, about 0.5,
about 0.6, about 0.7, about 0.75, about 0.8, about 0.9, about 1 ug
daily, about 1.5, about 2, about 2.5, about 3, about 4, about 5,
about 6, about 7, about 7.5, about 8, about 9, about 10, about 15,
about 20, about 25, about 30, about 40, about 50, about 60, about
70, about 75, about 80, about 90, or about 100 microgram (.mu.g)
daily, or any range thereof.
[0117] For example, an agent, such as, for example, bosentan or
ambrisentan, or a derivative thereof, may be administered at a
dosage of about 0.1, about 0.2, about 0.25, about 0.3, about 0.4,
about 0.5, about 0.6, about 0.7, about 0.75, about 0.8, about 0.9,
about 1, about 1.25, about 2, about 2.5, about 3, about 4, about 5,
about 6.25, about 10, about 12.5, about 20, about 25, about 30,
about 40, about 50, or about 62.5 mg daily, or any range
thereof.
[0118] For example, an agent, such as, for example, bosentan or
ambrisentan, or a derivative thereof, may be administered at a
dosage of less than about 0.1, about 0.2, about 0.25, about 0.4,
about 0.5, about 0.6, about 0.7, about 0.75, about 0.8, about 0.9,
about 1 ug daily, about 1.5, about 2, about 2.5, about 3, about 4,
about 5, about 6, about 7, about 7.5, about 8, about 9, about 10,
about 15, about 20, about 25, about 30, about 40, about 50, about
60, about 70, about 75, about 80, about 90, or about 100 microgram
(.mu.g) daily.
[0119] For example, an agent, such as, for example, bosentan or
ambrisentan, or a derivative thereof, may be administered at a
dosage of less than about 0.1, about 0.2, about 0.25, about 0.3,
about 0.4, about 0.5, about 0.6, about 0.7, about 0.75, about 0.8,
about 0.9, about 1, about 1.25, about 2, about 2.5, about 3, about
4, about 5, about 6.25, about 10, about 12.5, about 20, about 25,
about 30, about 40, about 50, or about 62.5 mg daily.
[0120] In some applications, administration on agent of the present
disclosure may be at a dosage greater than the accepted dosage for
previously known applications for the treatment of pulmonary
arterial hypertension (PAH). For example, an agent, such as, for
example, bosentan or a derivative thereof may be administered at a
dosage of about 250 mg/day or more, about 300 mg/day or more, about
450 mg/day or more, about 500 mg/day or more, about 600 mg/day or
more, about 750 mg/day or more, about 1000 mg/day or more, about
1500 mg/day or more, about 2000 mg/day or more, or about 2500
mg/day or more. For example, an agent such as ambrisentan or a
derivative thereof may be administered at a dosage of about 10
mg/day or more, about 12 mg/day or more, about 15 mg/day or more,
about 20 mg/day or more, about 25 mg/day or more, about 30 mg/day
or more, about 40 mg/day or more, about 50 mg/day or more, or about
100 mg/day or more.
[0121] In some therapeutic embodiments, an "effective amount" of an
agent is an amount that results in a reduction of at least one
pathological parameter. Thus, for example, in some aspects of the
present disclosure, an effective amount is an amount that is
effective to achieve a reduction of at least about 10%, at least
about 15%, at least about 20%, or at least about 25%, at least
about 30%, at least about 35%, at least about 40%, at least about
45%, at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, or at least
about 95%, compared to the expected reduction in the parameter in
an individual not treated with the agent.
[0122] As used herein, the term "subject" includes, but is not
limited to, humans and non-human vertebrates. In preferred
embodiments, a subject is a mammal, particularly a human. A subject
may be an individual. A subject may be an "individual," "patient,"
or "host." In some aspects, a subject is an individual diagnosed
with Alport syndrome. Diagnosis may be by any of a variety of
means, including, but not limited to, family history, clinical
presentation, pathological determination, and/or genetic testing.
Such as subject may be a male or a female. Non-human vertebrates
include livestock animals, companion animals, and laboratory
animals. Non-human subjects also include non-human primates as well
as rodents, such as, but not limited to, a rat or a mouse.
Non-human subjects also include, without limitation, chickens,
horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink,
and rabbits.
[0123] As used herein "in vitro" is in cell culture and "in vivo"
is within the body of a subject.
[0124] As used herein, "isolated" refers to material that has been
either removed from its natural environment (e.g., the natural
environment if it is naturally occurring), produced using
recombinant techniques, or chemically or enzymatically synthesized,
and thus is altered "by the hand of man" from its natural
state.
[0125] Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, and so forth used in
the specification and claims are to be understood as being modified
in all instances by the term "about." Accordingly, unless otherwise
indicated to the contrary, the numerical parameters set forth in
the specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques.
[0126] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0127] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. All numerical values, however,
inherently contain a range necessarily resulting from the standard
deviation found in their respective testing measurements.
[0128] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0129] The term "and/or" means one or all of the listed elements or
a combination of any two or more of the listed elements.
[0130] The words "preferred" and "preferably" refer to embodiments
of the invention that may afford certain benefits, under certain
circumstances. However, other embodiments may also be preferred,
under the same or other circumstances. Furthermore, the recitation
of one or more preferred embodiments does not imply that other
embodiments are not useful, and is not intended to exclude other
embodiments from the scope of the invention.
[0131] The terms "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0132] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
[0133] For any method disclosed herein that includes discrete
steps, the steps may be conducted in any feasible order. And, as
appropriate, any combination of two or more steps may be conducted
simultaneously.
[0134] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
[0135] The above discussion of the present invention is not
intended to describe each disclosed embodiment or every
implementation of the present invention. The description that
follows more particularly exemplifies illustrative embodiments. In
several places throughout the application, guidance is provided
through lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive
list.
[0136] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
.alpha.1.beta.1 Integrin-Mediated Rac1/CDC42-Dependent Mesangial
Process Invasion of Glomerular Capillary Tufts in Alport
Syndrome
[0137] With this example, a comparative analysis of glomerular
disease progression in Alport mice and CD151 knockout mice revealed
a progressive irregular deposition of mesangial laminin 211 in the
GBM. Co-localization studies showed that the mesangial integrin
.alpha.8.beta.1 also progressively accumulates in the capillary
loops of both models as well as in human Alport glomeruli,
indicating an invasion of the capillary loops by mesangial cell
processes. L-NAME salt-induced hypertension accelerated mesangial
cell process invasion and laminin 211 accumulation, suggesting
biomechanical strain plays a role in this mechanism. Cultured
mesangial cells showed reduced migratory potential when treated
with either integrin linked kinase inhibitor, Rac1 inhibitors,
CDC42 inhibitors, or by deletion of integrin .alpha.1.
Biomechanical stretching of cultured mesangial cells induced
pro-migratory cytokines TGF-.beta.1 and CTGF. Treatment of Alport
mice with a Rac1 inhibitor reduced mesangial cell process invasion
of the glomerular capillary tuft. Laminin .alpha.2-deficient Alport
mice show reduced mesangial process invasion, and laminin
.alpha.2-null cells show reduced migratory potential, indicating a
central role for mesangial laminins in progression of Alport
glomerular pathogenesis. Collectively, these findings predict a
role for biomechanical insult in the induction of mesangial cell
process invasion of the glomerular capillary tuft leading to the
irregular deposition of mesangial laminin 211 as an initiation
mechanism of Alport glomerular pathology.
[0138] The activation of genes encoding GBM matrix molecules,
matrix metalloproteinases (MMPs), and proinflammatory cytokines
have all been linked to the progression of Alport glomerular
disease. These, however, are events that occur after the onset of
proteinuria therefore downstream of disease initiation events
(Sayers et al., 1999, Kidney Int; 56(5):1662-1673; Cosgrove et al.,
2000, Am J Pathol; 157(5):1649-59; Rao et al., 2006, Am J Pathol;
169(1):32-46; Zeisberg et al., 2006, PLoS Medicine; 3(4), e100; and
Cosgrove et al., 2008, Am J Pathol; 172(3):761-7737-11). Consistent
with this notion, experiments aimed at blocking these pathways have
offered only limited therapeutic benefit in mouse models for Alport
syndrome (Cosgrove et al., 2000, Am J Pathol; 157(5):1649-59; Rao
et al., 2006, Am J Pathol; 169(1):32-46; Zeisberg et al., 2006,
PLoS Medicine; 3(4), e100; and Koepke et al., 2007, Nephrol Dial
Transplant; 22(4):1062-9). One of the earliest events is the
appearance of an irregular deposition of laminin 211 in the GBM of
Alport mice (Cosgrove et al., 2000, Am J Pathol; 157(5):1649-59),
an observation confirmed in both Alport dogs and human patients
with the disease (Kashtan et al., 2001, J Am Soc Nephrol;
12:252-60). This laminin is normally found only in the mesangium of
the glomerulus, and is not expressed in the GBM at any stage of
embryonic development (Miner et al., 1997, J Cell Biol;
137(3):685-701). Indeed several other mesangial matrix proteins
appear in the GBM of Alport mice, including laminin 111 and
fibronectin (Cosgrove et al., 1996, Genes Dev; 10(23): 2981-2992;
and St John and Abrahamson, 2001, Kidney Int; 60(3):1037-1046).
[0139] In the Alport glomerulus, the podocytes are exposed to GBM
that has an embryonic type IV collagen composition (Kalluri et al.,
1997, J Clin Invest; 99(10):2470-2478; and Harvey et al., 1998,
Kidney Int; 54(6):1857-1866). This could result in altered cell
signaling that may trigger the onset of the disease. It has been
proposed this type of mechanism may account for the reactivation of
laminin 111 expression in podocytes (Abrahamson et al., 2003,
Kidney Int; 63:826-34), a laminin found in the GBM during
development (Miner et al., 1997, J Cell Biol; 137(3):685-701).
Since the .alpha.1(IV)/.alpha.2(IV) collagen network contains
significantly fewer interchain disulfide crosslinks (Gunwar et al.,
1998, J Biol Chem; 273(15):8767-75), and since the Alport GBM is
thinner than normal (Kamenetsky et al., 2010, J Digital Imaging;
23:463-474) the Alport GBM is likely to be more elastic, resulting
in elevated biomechanical strain on the glomerular cells at their
points of contact the GBM. Consistent with this, glomeruli from
Alport mice have been shown to have elevated deformability relative
to wild type glomeruli (Wyss et al., 2011, Am J Physiol Cell
Physiol; 300:C397-C405), and salt-induced hypertension has been
shown to accelerate glomerular disease progression in Alport mice
(Meehan et al., 2009, Kidney Int; 76:968-976).
[0140] This example shows that deletion of laminin 211 in Alport
mice ameliorates the mesangial process invasion of the glomerular
capillary loops in Alport mice, demonstrating for the first time a
functional role for GBM laminin 211 in Alport glomerular
pathogenesis. The cellular origin of GBM laminin 211 has not been
previously determined. This example shows that the source of GBM
laminin 211 in Alport GBM is mesangial cell processes, which are
invading the capillary tufts. Salt-mediated hypertension
exacerbates this mesangial process invasion. A knockout mouse for
the integrin .alpha.3.beta.1 co-receptor CD151, which results in
reduced adhesion of podocytes pedicles to GBM laminin 521, also
develops mesangial process invasion of the capillary loops with GBM
deposition of laminin 211, demonstrating the same phenotype for a
completely unrelated component of the capillary structural barrier.
The CD151 knockout mouse model also shows accelerated glomerular
disease progression in response to hypertension (Sachs et al.,
2012, J Clin Invest; 122(1):348-58). Mesangial cell culture studies
show that biomechanical stretching induces pro-migratory cytokines
TGF-.beta.1 and CTGF, both known to be induced in Alport glomeruli
(Sayers et al., 1999, Kidney Int; 56(5):1662-1673; and Koepke et
al., 2007, Nephrol Dial Transplant; 22(4): 1062-9). Inhibitor
studies indicate that migration is mediated through .alpha.1.beta.1
integrin signaling through the Rho GTPases RAC1 and CDC42.
Consistent with this, .alpha.1 integrin deletion in Alport mice was
previously shown to ameliorate glomerular disease progression and
slow the accumulation of laminin 211 in Alport GBM (Cosgrove et
al., 2000, Am J Pathol; 157(5):1649-59). This example shows that
mesangial process invasion of the capillary loops is ameliorated in
.alpha.1 integrin null Alport mice. These data define a surprising
role for biomechanical strain mediated-induction of mesangial cell
process invasion as a key aspect of Alport glomerular disease
initiation, and identify novel therapeutic targets to blocking this
process.
Results
[0141] GBM laminin 211 in Alport mice is of mesangial origin. In
the glomerulus, laminin 211 is normally found only in the mesangial
matrix. FIG. 1 (A-C) demonstrates mesangial distribution of laminin
211 in wild type mice, which is distinct from the glomerular
basement membrane (collagen .alpha.3(IV)). In Alport glomeruli,
FIG. 1 (D-F) demonstrates the irregular distribution of laminin 211
in the GBM which appears to accumulate preferentially in
irregularly thickened regions of the GBM (here the GBM is marked by
immunostaining with antibodies specific for laminin .alpha.5). The
cellular source of the GBM laminin 211 has never been determined.
Dual immunofluorescence labeling with antibodies against laminin
.alpha.2 and integrin .alpha.8 show mesangial specific
immunostaining in wild type glomeruli (FIG. 2 (A-C)), as reported
previously (Hartner et al., 1999, Kidney Int; 56(4):1468-80). In
Alport glomeruli (at 7 weeks of age) immunostaining for both
laminin .alpha.2 and integrin .alpha.8 appears to have spread into
the capillary loops consistent with a mesangial cell process
invasion of the capillary loops (FIG. 2 (D-F)). Dual
immunofluorescence immunostaining using the basement membrane
marker laminin .alpha.5 with the mesangial marker integrin .alpha.8
confirms that integrin .alpha.8 immunostaining, while absent from
the GBM in wild type mice (FIG. 2 (G-I)), is clearly present in
most of the GBM of Alport mice (FIG. 2 (J-L)). Collectively these
data indicate that GBM laminin 211 arises from a mesangial cell
process invasion of the capillary loops, and thus is of mesangial
cell origin.
[0142] To determine the relevance of this observation to human
Alport syndrome cryosections from human Alport necropsy kidney
sections were stained with antibodies specific for integrin
.alpha.8 and laminin .alpha.5. The results in FIG. 3 (A-C) show
that mesangial processes are clearly present adjacent to the
laminin .alpha.5-immunopisitive GBM in the human specimen.
[0143] Mesangial process invasion of the capillary loops is
exacerbated by elevated biomechanical strain. An earlier report
demonstrated that hypertension exacerbated the progression of
Alport glomerular disease (Meehan et al., 2009, Kidney Int;
76:968-976). Hypertension accelerated several aspects of glomerular
disease progression including proteinuria and induction of matrix
metalloproteinases. The accumulation of GBM laminin 211 was also
accelerated. As shown in FIG. 4 (A-F), salt-induced hypertension
clearly accelerates the inundation of the glomerular capillary
loops by mesangial processes as evidenced by the presence of
integrin .alpha.8 immunopositivity in the GBM (FIG. 4(D-F)).
[0144] It is likely the increased biomechanical stress on the
glomerular capillary tuft in Alport glomeruli is due to the change
in GBM type IV composition from dual networks of
.alpha.1(IV)/.alpha.2(IV) and
.alpha.3(IV)/.alpha.4(IV)/.alpha.5(IV) collagen to one comprised
only of .alpha.1(IV)/.alpha.2(IV) collagen. The latter is thinner
and known to contain fewer interchain disulfide crosslinks (Gunwar
et al., 1998, J Biol Chem; 273(15):8767-75) which would intuitively
be expected to result in increasing the elasticity of the
glomerular filtration barrier. In order to provide independent
validation, a completely different model was examined that would
also be expected to affect the elastic integrity of the glomerular
filtration barrier, the CD151 knockout mouse. CD151 is a
tetraspanin co-receptor for integrin .alpha.3.beta.1 which
functions to increase the affinity of integrin .alpha.3.beta.1 for
its GBM ligand, laminin .alpha.5 (Nishiuchi et al., 2005, Proc Natl
Acad Sci USA; 102(6): 1939-44). Deletion of CD151 results in
glomerular disease with morphological changes in the GBM strikingly
similar to Alport syndrome (Baleato et al., 2008, Am J Pathol;
173(4):927-37). Recently it was shown that hypertension accelerates
the progression of glomerular disease in the CD151 knockout mouse,
similar to our observations for the Alport mouse (Sachs et al.,
2012, J Clin Invest; 122(1):348-58). Considering all of this,
glomeruli from the CD151 knockout mouse were examined for mesangial
process invasion and laminin 211 deposition in the GBM. The results
in FIG. 5 are impressive, in that this mouse shows a near complete
inundation of the glomerular capillary tufts with integrin .alpha.8
and laminin .alpha.2 immunopositivity, demonstrating mesangial
process invasion and deposition of mesangial laminins in the GBM in
this genetically unrelated model.
[0145] If biomechanical strain can induce the activation of
mesangial process invasion of the capillary tuft, pro-migratory
responses will be activated in vitro by mechanically stretching
cultured primary mesangial cells. Primary cultured mesangial cells,
derived from 129 Sv/J mice, were subjected to cyclic cell
stretching using the Flexcell system for 24 hours. Expression of
several pro-migratory cytokines was quantified by real time RT-PCR.
The results in FIG. 6 demonstrate that expression of both
TGF-.beta.1 and CTGF are significantly elevated in cells subjected
to biomechanical stretching relative to cells cultured under
identical conditions, but not subjected to stretch.
[0146] Mesangial cell migration (in vitro) and mesangial process
invasion of the glomerular capillary loops (in vivo) are regulated
by integrin .alpha.1.beta.1 mediated Rac1/CDC42 crosstalk. Earlier
work demonstrated that deletion of .alpha.1 integrin markedly
attenuated the progression of glomerular disease in Alport mice
(Cosgrove et al., 2000, Am J Pathol; 157(5):1649-59). Although is
highly likely that disease attenuation in integrin .alpha.1-null
Alport mice emanates from the mesangial compartment where integrin
.alpha.1.beta.1 is highly expressed, the molecular mechanism
underlying this effect has remained unclear. FIG. 7 shows that
deletion of .alpha.1 integrin markedly reduces the dynamics of
mesangial process invasion of the capillary tufts in Alport mice,
consistent with the reduction in GBM laminin 211 deposition shown
here and previously (Cosgrove et al., 2000, Am J Pathol;
157(5):1649-59).
[0147] Since it is well established that the formation of filopodia
and lamellipodia require the concerted action of the small GTPases
Rac1 and CDC42 (Vicente-Manzanares et al., 2009, J Cell Sci;
122(2):199-206), cell migration assays were performed using the
Boyden chamber approach to determine whether such a functional
connection was evident in cultured wild type and integrin
.alpha.1-null mesangial cells. The results in FIG. 8 (A) show that
integrin .alpha.1-null mesangial cells show a significant reduction
in migratory potential relative to wild type mesangial cells.
Migration of wild type cells was significantly reduced when cells
were treated with either the integrin linked kinase inhibitor
QLT-0267, the Rac1 inhibitor NSC 23766, or the CDC42 inhibitor
ML141. Cell migration of wild type cells were not affected by
treatment with the pan AKT inhibitor GSK 690693. Integrin
.alpha.1-null mesangial cell migration was significantly reduced
when cells were treated with ILK inhibitors, but unaffected when
treated with Rac1 inhibitors, demonstrating that deletion of
.alpha.1 integrin abrogates Rac1-dependent cell migration.
[0148] Treatment of cells with the bacterial endotoxin
lipopolysaccharide (LPS) activates both Rac1 and CDC42 GTPases
(Sanlioglu et al., 2001, J Biol Chem; 276(32):30188-98; and Fessler
et al., 2004, J Biol Chem; 279(38):39989-98), and is known to
induce the formation of both lamellipodia and filopodia in cultured
mesangial cells (Bursten et al., 1991, Am J Pathol; 139(2):371-82).
Cultured wild type mesangial cells were treated with LPS, the actin
filaments stained with phalloidin, and the cultures examined for
morphological changes. As shown in FIG. 8 (B), after 30 minutes,
treated cells undergo a stark morphological change about half of
the cells sprouting numerous filopodia (denoted by asterisks), that
are easily discernable, blinded, in numerous replicate experiments.
Cells treated with LPS in combination with either the Rac1
inhibitor NSC 23766 or the CDC42 inhibitor ML 141 could not be
distinguished in blinded experiments form untreated wild type
mesangial cells (FIG. 8 (B) panels C and D, respectively).
[0149] Interestingly, treatment of integrin .alpha.1-null mesangial
cells with LPS had no discernable effect on cell morphology. To
further validate these findings, either wild type or .alpha.1-null
mesangial cell cultures were stimulated with LPS in the presence or
absence of either Rac1 or CDC42 inhibitors and performed
immunofluorescence analysis for CDC42 localization and pull down
assays for activated Rac1. As shown in FIG. 8 (C), treatment of
cells with LPS resulted in polarized localization of CDC42
associated with staining in adjacent filopodia (panel B insert), an
established characteristic of CDC42 activation (Etienne-Manneville
and Hall, 2001, Cell; 106:489-498; and Huang et al., 2011, J Cell
Biochem; 112(6):1572-1584). Treatment of these cells with Rac1
inhibitor abolished this polarized activation, indicating
cross-talk between Rac1 and CDC42. Integrin .alpha.1-null mesangial
cells did not respond to LPS activation with polarized CDC42
localization. Pull down assays demonstrate that LPS treatment does
indeed activate Rac1, and that pre-treatment of cells with the Rac1
inhibitor abolishes its activation (FIG. 8C(d)). Interestingly,
pre-treatment of cells with CDC42 inhibitors did not block LPS
mediated Rac1 activation, suggesting that, while Rac1 inhibitors
block LPS-CDC42 activation (FIG. 8 (C), panel C), CDC42 inhibitors
do not block Rac1 activation (FIG. 8 (C), panel D).
[0150] To examine the effect of Rac1 inhibitors on Alport
glomerular disease progression, either wild type or Alport mice
were treated with inhibitors by IP injection from 2 weeks to 6
weeks of age. Glomeruli were examined for mesangial process
invasion of the capillary tufts by dual immunofluorescence
microscopy using antibodies specific for either integrin .alpha.8
or the GBM marker laminin .alpha.5. The results in FIG. 9 (A-F)
demonstrate that saline-injected mice show significant
co-localization of integrin .alpha.8 and laminin .alpha.5
throughout many of the glomerular capillary tufts, while mice
injected with the Rac1 inhibitor showed very little mesangial
process invasion. Combined, the data in FIG. 7, FIG. 8, and FIG. 9
confirm that mesangial process invasion of the glomerular
capillaries is a Rac1-dependent process, and that Rac1 activation
is attenuated by integrin .alpha.1 deletion both in vitro and in
vivo. Furthermore, LPS activation of filopodia in wild type
mesangial cells (but not in .alpha.1-null mesangial cells) involves
both Rac1 and CDC42 activation, suggesting .alpha.1.beta.1
integrin-dependent cross talk between the two small GTPases in the
signaling complex.
[0151] Laminin 211 enhances mesangial cell migration and mesangial
process invasion of the capillary loops. In a related study to
determine whether GBM laminin 211 contributed mechanistically to
the progression of Alport glomerular disease, a laminin
.alpha.2-deficient mouse was crossed with the Alport mouse to
produce a double knockout. One effect of laminin .alpha.2
deficiency was a marked reduction of mesangial process invasion of
the capillary loops (FIG. 10 (A)). This indicates that laminin 211
might facilitate mesangial process invasion of the capillary loops.
Thus, cell migration assays were performed on either laminin 211 or
laminin 521 (GBM laminin). Two different laminin preparations were
used. One was extracted laminin from either placenta (primarily
laminin 511) or muscle (primarily laminin 211); the other
commercially available purified recombinant laminin heterotrimers.
A scratch wound assay was used as opposed to the Boyden chamber, to
determine the role of specific extracellular matrix in potentiating
mesangial cell migration. As shown in FIG. 10 (B), wild type
mesangial cells migrate much more efficiently on laminin 211
compared to laminin 521. While the effect was more pronounced on
the muscle laminin preparation relative to the placental laminin
preparation, it is also clear on the pure recombinant laminin
substrates. To more directly confirm the role of laminin .alpha.2
in migratory potential, the relative migration of wild type
mesangial cells to mesangial cells derived from laminin
.alpha.2-deficient mice was measured, this time using the Boyden
chamber approach. The results in FIG. 10 (C) represent multiple
derivations of both cell types, and demonstrate a statistically
significant reduction in the migratory potential of laminin
.alpha.2-deficient mesangial cells relative to wild type mesangial
cells. Collectively the data in FIG. 10 indicate that laminin 211
deposition by the mesangial processes functionally contributes to
the process invasion of the capillary tuft in Alport and
CD151-knockout glomeruli.
Discussion
[0152] Earlier studies of Alport mouse, dog, and humans reported
the presence of "abnormal" laminins in the GBM, including laminin
211 and laminin 111 (Cosgrove et al., 2000, Am J Pathol;
157(5):1649-59; Kashtan et al., 2001, J Am Soc Nephrol; 12:252-60;
and Abrahamson et al., 2003, Kidney Int; 63:826-34). These laminins
tend to accumulate in areas of irregular thickening of the GBM, and
these thickened areas have been shown to be more permeable to
ferritin, suggesting that they are comprised of loosely assembled
or partially degraded extracellular matrix (Abrahamson et al.,
2007, J Am Soc Nephrol; 18:2465-72). In addition to the "abnormal"
laminins, fibronectin and heparin sulfate proteoglycans have also
been reported to accumulate in the GBM of Alport mice (Cosgrove et
al., 1996, Genes Dev; 10(23): 2981-2992). What all of these ECM
components have in common is that they are predominantly found in
the mesangial matrix (Schlondorff and Banas, 2009, J Am Soc
Nephrol; 20:1179-87).
[0153] This example determined that these abnormal GBM matrix
molecules that progressively accumulate in the Alport GBM are of
mesangial cell origin. Integrin .alpha.8 was used as a specific
mesangial cell surface marker to demonstrate that mesangial
processes invade the capillary tufts and co-localize with laminin
211, a mesangial laminin. Integrin .alpha.8 is expressed in
mesangial cells, but not in other glomerular cell types (Hartner et
al., 1999, Kidney Int; 56(4):1468-80), and its expression is
generally restricted to smooth muscle cells and neuronal cell types
(Bossy et al., 1991, EMBO J; 10(9):2375-2385; and Schnapp et al.,
1995, J Cell Sci; 108:537-544). Mesangial process invasion of the
glomerular capillary tufts was exacerbated by hypertension,
indicating that the mechanism triggering this event was mediated by
biomechanical stress, likely at the interface between the mesangial
processes and the sub-endothelial interface with the glomerular
capillaries, an area known to provide important structural support
for the capillary loops (Schlondorff and Banas, 2009, J Am Soc
Nephrol; 20:1179-87). The Alport mutations, which can be either
autosomal recessive (mutations in either COL4A3 or COL4A4 genes
(Mochizuki et al., 1994, Nat Genet; 8(1):77-81)) or X-linked
(mutations in COL4A5 (Barker et al., 1990, Science; 248(4960):
1224-7)) result in the absence of the collagen
.alpha.3(IV)/.alpha.4(IV)/.alpha.5(IV) network from the GBM. The
consequence is a thinner GBM comprised of only .alpha.1(IV) and
.alpha.2(IV) collagens, which have been shown to contain fewer
interchain disulfide crosslinks (Gunwar et al., 1998, J Biol Chem;
273(15):8767-75). This structural change alters the biomechanical
properties of the capillary tuft, resulting in stresses on the
cells comprising the tuft even under normal glomerular blood
pressures.
[0154] A second model was examined, the CD151 knockout mouse, which
would also show enhanced strain on the capillary tufts. In this
model, enhanced strain arises as a result of reduced adhesion of
the podocyte pedicles to the GBM due to reduced affinity for the
podocyte integrin .alpha.3.beta.1 for its GBM ligand laminin 521
(Nishiuchi et al., 2005, Proc Natl Acad Sci USA; 102(6): 1939-44).
Mesangial process invasion of the glomerular capillary tufts in the
CD151 mouse was even more robust than that for the Alport model.
Like the Alport model (Meehan et al., 2009, Kidney Int;
76:968-976), glomerular pathology in the CD151mouse model, which
shows ultrastructural lesions in the GBM strikingly similar to
Alport syndrome (Baleato et al., 2008, Am J Pathol; 173(4):927-37;
and Sachs et al., 2006, J Cell Biol; 175(1):33-9) is significantly
exacerbated under hypertensive conditions (Sachs et al., 2012, J
Clin Invest; 122(1):348-58). Collectively this evidence supports
the notion that mutations affecting structural integrity of the
glomerular capillary tuft result in unnatural stresses on the cells
comprising the tuft. In the mesangial cell compartment this results
in mesangial cell invasion of the tuft and deposition of matrix
proteins in the GBM that are of mesangial cell origin.
[0155] Earlier work showed that deletion of the mesangial integrin
all 1 in Alport mice resulted in a marked attenuation in the
progression of the glomerular pathology, with reduced proteinuria
and a near doubling of lifespan (Cosgrove et al., 2000, Am J
Pathol; 157(5):1649-59). The mechanism underlying the influence of
mesangial .alpha.1.beta.1 integrin on Alport renal disease
progression has, until the present example, remained unclear. This
example shows that mesangial process invasion is markedly
attenuated in integrin .alpha.1-null Alport mice relative to
strain/age matched Alport mice. This indicates that the signaling
pathway that activates actin cytoskeletal rearrangements is
perturbed in the absence of .alpha.1.beta.1 integrin. Further,
decreased migratory potential was observed for primary cultures of
.alpha.1-null mesangial cells relative to wild type mesangial cells
from strain/age matched mice (FIG. 8 (A)).
[0156] Lipopolysaccharide, which activates both Rac1 and CDC42 in
wild type mesangial cells, failed to activate Rac1 or CDC42 (FIG. 8
(B)), and failed to activate actin cytoskeletal rearrangements in
cultured .alpha.1-null mesangial cells. Collectively these data
explain why deletion of .alpha.1-integrin results in attenuation of
Alport glomerular pathogenesis and indicate that .alpha.1.beta.1
integrin is a key sensor of biomechanical strain at the glomerular
capillary tuft and participates in the adhesive signaling mechanism
that links to the Rho GTPases Rac1 and CDC42 which activate actin
polymerization dynamics required to process invasion of the
glomerular capillary tufts.
[0157] Classically, Rac1 activation is associated with lamellipodia
formation and CDC42 activation is associated with filopodia
formation (Nobes and Hall, 1995, Cell; 81(1):53-62).
[0158] Recently, evidence for crosstalk between the two Rho GTPases
has emerged (Zamudio-Meza et al., 2009, J Gen Virol; 90(Pt
12):2902-11). This phenomenon is likely regulated through the
guanine nucleotide exchange factor .beta.1pix, which contains
binding sites for both CDC42 and Rac1 (Chahdi et al., 2004, Biochem
Biophys Res Commun; 317(2):384-9; and Chahdi et al., 2005, J Biol
Chem; 280(1):578-84). This example provides evidence for cross-talk
between Rac1 and CDC42 in cultured mesangial cells regulating actin
cytoskeletal rearrangement including: showing that treatment of
mesangial cells with LPS, known to activate rapid actin
cytoskeletal rearrangement (Bursten et al., 1991, Am J Pathol;
139(2):371-82), activates Rac1 in wild type mesangial cells (FIG. 8
(C), panel D); showing that membrane localization of CDC42, a known
prerequisite for its activation, is blocked by addition of RAC1
inhibitors coincident with LPS stimulation (FIG. 8 (C), panels
A-C); and showing that inclusion of either Rac1 inhibitors or CDC42
inhibitors upon stimulation of mesangial cell cultures with LPS
blocks actin cytoskeletal rearrangements (FIG. 8 (B)).
[0159] Mesangial cell cultures subjected to cyclic biomechanical
strain expressed elevated levels of the pro-migratory cytokines
CTGF and TGF-.beta.1, providing further evidence that biomechanical
strain activates actin cytoskeletal dynamics required for mesangial
process invasion. Both CTGF and TGF-.beta.1 signaling have been
shown to activate CDC42 (Edlund S et al., 2002, Mol Bio Cell;
13:902-14; and Crean et al., 2004, FASEB J; 18(13):1541-3), and
both cytokines have been shown to be induced in Alport glomeruli
(Sayers et al., 1999, Kidney Int; 56(5):1662-1673; and Kashtan et
al., 2001, J Am Soc Nephrol; 12:252-60) indicating that activation
of these signaling pathways might be an important underlying
mechanism for the activation of mesangial process invasion of
glomerular capillary tufts in Alport syndrome. Indeed, earlier work
showed that inhibition of TGF-.beta.1 in the Alport mouse resulted
in abrogation of GBM thickening, in support of this notion
(Cosgrove et al., 2000, Am J Pathol; 157(5):1649-59). And, when
TGF-.beta.1 was inhibited in .alpha.1 integrin-null Alport mice, a
synergistic improvement in glomerular disease was observed
suggesting that TGF-.beta.1 and integrin .alpha.1 are working
through distinct pathways (Cosgrove et al., 2000, Am J Pathol;
157(5):1649-59). Based on the current study, these pathways may
converge on strain-mediated activation of Rac1/CDC42 in the
mesangial cell compartment.
[0160] While the deposition of laminin 211 in the GBM of Alport
mice was described more than 10 years ago (Cosgrove et al., 2000,
Am J Pathol; 157(5):1649-59; and Kashtan et al., 2001, J Am Soc
Nephrol; 12:252-60), a functional role for this laminin in Alport
glomerular pathology has not been described. This example shows
reduced mesangial process invasion of the glomerular capillary
loops in Alport mice that are also lacking laminin .alpha.2,
indicating that laminin 211 itself promotes the migration of
processes into the glomerular capillary loops (FIG. 9 (A)).
Consistent with this, the example shows that wild type mesangial
cells migrate more robustly when cultured on laminin 211 compared
to laminin 521, and that primary mesangial cells from laminin
.alpha.2-deficient mice show impaired migration relative primary
wild type mesangial cells from age/strain matched mice (FIG. 9
(B-C)). While modulation of mesangial cell migration by ECM has
been described previously (Person et al., 1988, Am J Pathol;
133(3):609-14), this example shows that the strain-mediated
mesangial process invasion of the capillary loops is enhanced by
mesangial cell secreted laminin 211, which may explain why laminin
211 accumulates in the patchy irregularly thickened regions of the
Alport GBM (see FIG. 1).
[0161] This example shows that the changes in the biophysical
properties of the Alport glomerular capillary tuft results in
biomechanical stresses that result in the induction of pathologic
processes. Parallel observations in Alport and CD151 mouse models,
including mesangial process invasion of the glomerular capillary
tufts and deposition of laminin 211 provide additional support,
since the two mouse models arise from mutations that would be
expected to relax the structural integrity of the glomerular
capillary tufts, but are otherwise mechanistically unrelated to
each other. Recent studies of the biophysical properties of Alport
glomeruli from pre-proteinuric mice reported increased
deformability and suggested the glomeruli were "squishy" (Wyss et
al., 2011, Am J Physiol Cell Physiol; 300:C397-C405). Collectively,
this example supports a model where biomechanical stresses on the
glomerular capillary tufts activate a promigratory signaling
cascade in mesangial cells involving integrin
.alpha.1.beta.1-mediated activation of Rac1/CDC42 crosstalk. This
activation culminates in the invasion of the capillary loops by
mesangial processes. These processes clearly deposit laminin 211,
which further exacerbates the mesangial process invasion. In
addition to laminin 211, other mesangial matrix molecules are
likely deposited in the GBM, and local action of mesangial
cytokines (TGF-.beta.1 and CTGF, for example) and MMPs might also
contribute to the structural and functional properties of the
Alport GBM (irregular thickening, splitting, permeability, etc.).
In addition, all of these events are very likely to influence
podocyte cell health. Thus, mesangial process invasion of the GBM
is an important early event that precipitates glomerulosclerosis in
Alport syndrome. The observation of mesangial process invasion of
glomerular capillary loops in human Alport glomeruli provides
relevance for these observations to the human disease. A better
understanding of the activation process might reveal novel targets
capable of preventing this event and arresting the Alport
glomerular pathogenesis in its pre-initiated state.
Methods
[0162] Mice. All mice used in these studies were on pure genetic
backgrounds. Autosomal recessive Alport mice were on the 129 Sv
background. X-linked Alport mice were on the C57 Bl/6 background,
laminin .alpha.2-deficient mice were on the 129 Sv background,
integrin .alpha.1-null mice were on the 129 Sv background (Gardner
et al., 1996, Dev Biol; 175(2):301-13), and CD151 knockout mice
were on the FVB background (Takeda et al., 2007, Blood;
109(4):1524-32). All experiments were performed using
strain/age-matched control mice. All animal studies were conducted
in accordance to USDA approved standards and under the approval of
the institutional IACUC. Every effort was made to minimize pain and
discomfort.
[0163] Immunofluorescence microscopy. Fresh frozen kidneys were
sectioned at 8 m and acetone fixed. Sections were incubated
overnight at 4.degree. C. with 0.3% PBST (Triton X-100), 5% Fetal
Bovine Serum, and with two of the following antibodies: rat
anti-mouse Laminin-2 antibody (Sigma) at 1:200, goat anti-mouse
Integrin .alpha.8 antibody (R & D Systems) at 1:100, rabbit
anti-mouse Laminin-5 antibody at 1:200, rabbit anti-human Laminin-5
antibody (GeneTex) at 1:500, rabbit anti-mouse CDC42 antibody
(ProteinTech) at 1:50, and goat anti-mouse .alpha.-actinin-4
antibody (Santa Cruz) at 1:50. Affinity purified rabbit
anti-collagen .alpha.3(IV) antibodies were as previously described.
Slides were rinsed with 1.times.PBS and incubated with the
appropriate Alexa Fluor donkey secondary antibodies at 1:300 for 1
hour at room temperature. They were then rinsed again with
1.times.PBS and mounted with Vectashield Mounting Medium with Dapi
(Vector).
[0164] MES Migration (insert). Transwell cell migration assays were
performed as described by Daniel et al. (Daniel et al., 2012, Lab
Invest; 92(6): 812-26) with some modifications. 8 micron, 24-well
plate control inserts (BD Bioscience, Bedford, Mass.) were coated
overnight at 4.degree. C. with 100 .mu.l of 0.1% gelatin/PBS then
washed 1.times. with PBS. MES cultures were incubated in 1% FCS
overnight, then 0.05% BSA-containing media for at least 8 hours,
washed 1.times. with PBS and carefully tryspinized to ensure a
single cell suspension and limited "clumping" of cells. After
serum-neutralization and subsequent centrifugation, .about.100,000
cells were resuspended in 1.5 mls of 0.05% BSA media containing
activators/inhibitors. The wells of a 24-well plate were filled
with 0.75 mls of 10% FCS-containing media plus
activators/inhibitors (excluding 0.05% BSA control well). 0.5 ml of
cell-suspension was loaded into the gelatin-coated insert and the
insert placed in a well. Wells were visually inspected for bubbles
beneath insert and equal distribution of cell-suspension. Cells
were allowed to migrate overnight (.about.18 hrs). Using a
moistened cotton swab, non-migrated cells were liberated from the
apical-side of the insert by gentle but firm rubbing. A second swab
repeated the removal and was followed by a single wash with PBS.
Inserts were fixed, stained and washed (2.times.) in companion 24
well plate(s) containing 0.5 mls MEOH, 0.5 mls 1% Toluidine Blue in
1% Borax and 0.5 mls distilled H2O, respectively. Inserts were air
dried and counted at 100.times. magnification. Five fields were
counted on each insert including one center and four periphery
areas. Data was expressed as relative to 10% FCS control well (set
equal to one).
[0165] Scratch wound migration assay. For Basal Lamina studies
SUPERFROST.TM. Plus (VWR) microscope slides were coated with the
following: 100 ng/ml MEROSIN.TM. (Millipore), 100 ng/ml human
placental laminin (Sigma-Aldrich), 20 ng/ml human rlaminin-211
(BioLamina), or 20 ng/ml human rlaminin-521 (BioLamina) per
manufacturer's suggestion. Slide(s) were placed in a tissue culture
dish and an 8.times.8 mm cloning ring (Bellco Glass) placed on the
coated area. A 100 .mu.l of cell suspension (.about.30,000 cells)
in 1% FBS-containing media was added to the cloning ring and the
cells were allowed to attach for .about.8 hours, PBS was placed in
the dish and the ring removed. A .about.0.3-0.5 mm swath of cells
was removed was by running a serological pipette at a
.about.45.degree. angle through the monolayer. After capturing
images of removed cells, slides were incubated for 24 hours in 1%
FBS containing media, washed with PBS, fixed in methanol for 5
minutes, air dried and stained for 30 minutes with modified Giemsa
Stain (Sigma-Aldrich). Images of previously photographed fields
were captured using a Leica MZ10F Microscope fitted with a DFC310FX
camera.
[0166] Biomechanical stretching of cultured mesangial cells. Low
passage, sub confluent, primary mesangial cells were trypsinized
and seeded onto BIOFLEX.TM. 6-well plates (Flexcell International
Corp) coated with Rat tail type I collagen (BD Biosciences). Cells
were plated in 5% FCS containing media at densities that resulted
in 20-40% confluence. 0.5% FCS media was placed on the cells the
next day. 48 hours later the media was changed and the cultures
exposed to a regimen of 60 cycles of stretch and relaxation per
minute with amplitude of 10% radial surface elongation. The
Flexercell Strain Unit FX4000 (Flexcell International Corp.,
Hillsborough, N.C.) was used to induce stretch/relaxation for 18
hours according to manufacturer's directions. Cells grown
identically, but not exposed to stretch, served as controls.
[0167] Real time qRT-PCR. Total RNA was reverse transcribed using
SUPERSCRIPT.RTM. III (Invitrogen) with Oligo(dT).sub.20 Primer
(Invitrogen). The real time PCR was carried out using TAQMAN.RTM.
Gene Expression Master Mix (Applied Biosystems), and quantified
using ABI PRISM.RTM. 7000 sequence detection system (Applied
Biosystems). Samples were normalized to Mouse GAPDH Endogenous
Control VIC.RTM. Probe (Applied Biosystems catalogue #4352339E)
which was run alongside the CTGF and TGF.beta.-1 TAQMAN.RTM. Gene
Expression Assay Probes (Applied Biosystems catalogue #4331182).
Each of the samples were run in triplicate with a final reaction
volume of 50 .mu.l with the following cycling parameters:
50.degree. C. for 2 min, 95.degree. C. for 10 min, followed by 40
cycles of a two-step PCR consisting of 95.degree. C. for 15 s and
60.degree. C. for 1 min. Relative changes in gene expression were
determined by calculating the fold change using the comparative
C.sub.T method of 2.sup.-.DELTA..DELTA.CT.
[0168] Activation of mesangial cell cultures by treatment with LPS.
Sub-confluent mesangial cells were tryspinized; plated at low
density on Rat tail type 1 collagen (BD Biosciences) coated
cytology slides (VWR) and incubated overnight in 1% FCS-containing
media. One hour after the addition of serum-free media, 1 .mu.M
CDC42 Inhibitor (KSC-23-233) and 10 .mu.M Rac-1 Inhibitor,
NSC-23766 (Tocris) were added to individual slides and allowed to
incubate for an additional hour. 10 ng/ml Lipopolysaccharides
(Sigma-Aldrich) was added to cells, incubated 1 hour, fixed in ice
cold acetone for 5 minutes and allowed to air dry .about.2 hours.
Cells were stained with a 1:100 dilution of antibodies specific for
CDC42 (10155-1-AP, PROTEINTECH.TM.), and phalloidin (Molecular
Probes) imaged. Untreated, LPS alone and LPS plus inhibitors
treatments were repeated on two different derivations of primary
mesangial cells with qualitatively consistent results.
[0169] Pull down assay. Pull down experiments for Rac1 in mesangial
cells were done using the Rac1 Activation Assay Bicochem Kit
(BK035, Cytoskeleton Inc., CO) and according to manufacturer
instructions with minor modifications. Briefly, 500-800 .mu.g of
protein lysates were incubated with 20 .mu.l of PAK-PBD beads for 1
hour at 4.degree. C. Pull down samples and total protein lysates
(30-50 .mu.g of protein) were run in a 12% SDS-PAGE gel,
transferred to PVDF membranes and blocked in 5% milk for 30 minutes
at room temperature. Rac-1 antibody incubation was done overnight
at 4.degree. C. with rocking. After secondary antibody incubation
and several washes membranes were developed using the ECL Plus kit
(32134, Pierce, Ill.) pull-down experiments or the SuperSignal West
Femto kit (34094, Pierce, Ill.) for total lysates. Films were
exposed for 40 min and 5 min respectively and developed using a
film processor (Biomedical Imaging Systems, Model SRX-101A).
[0170] Confocal microscopy. Slides were cover slipped using
Vectashield mounting medium containing DAPI to counter-stain the
nuclei (Vector Lab, CA) and confocal images captured using a Zeiss
AxioPlan 2IF MOT microscope interfaced with a LSM510 META confocal
imaging system, using a 63.times. NA: 1.4 oil objective. Final
figures were assembled using Adobe Photoshop and Illustrator
software (Adobe Systems, CA).
[0171] This example has now published as ".alpha.1.beta.1
integrin/Rac1-dependent mesangial invasion of glomerular
capillaries in Alport syndrome," Zallocchi M, Johnson B M, Meehan D
T, Delimont D, Cosgrove D, Am J Pathol. 2013 October;
183(4):1269-80. doi: 10.1016/j.ajpath.2013.06.015. Epub 2013 Aug.
2, which is hereby incorporated by reference in its entirety.
Example 2
Endothelin Blockade with Bosentan Ameliorates Renal Pathology
[0172] This example describes a new etiology for Bosentan action in
Alport glomerular disease through its capacity to block endothelin
receptors on mesangial cells, blocking Rac1/CDC42 which prevents
mesangial invasion of the glomerular capillaries and thereby
ameliorates renal disease progression. This example substantiates,
for the first time, a new use for the known drug Bosentan.
[0173] One potential activator of cytoskeletal rearrangement in
mesangial cells is endothelin receptor mediated activation of
Rac1/CDC42. A significant amount of work has been done using
cultured mesangial cells that suggests a functional link between
endothelin receptor activation and activation of the rho GTPases,
Rac1 and CDC42 (reviewed in Sorokin, 2011, Contrib Nephrol;
172:50-62). As shown in Example 1 and herein, when Rac1 activation
was blocked, mesangial process invasion of glomerular capillaries
was ameliorated (see also Zallocchi et al., 2013, Am J Pathol;
183(4):1269-80).
[0174] To determine whether endothelin-1 expression is linked to
biomechanical strain on the glomerular capillary walls
pre-proteinuric X-linked Alport mice on the C57 Bl/6 background
mice were treated with Ramipril, a commercially available
angiotensin converting enzyme (ACE) inhibitor to make the animals
hypotensive. A second cohort of animals was given L-NAME salts to
make them hypertensive, and a third cohort was given drinking water
with no additives. The effect of these treatments on blood pressure
was confirmed directly using a CODA-2 tail cuff blood pressure
monitoring device specifically designed for mice. The effect of
these treatments was a significant elevation in blood pressures
(both systolic and diastolic) when comparing ramipril-treated mice
with salt-treated mice (FIG. 12). Kidney sections were dual stained
with antibodies specific for endothelin-1 and CD31 (a marker for
endothelial cells). Images of representative glomeruli are shown in
FIG. 11. Note the fact that endothelin-1 expression is barely
visible in glomeruli from Ramipril-treated mice (FIG. 11 (A)),
while readily visible in untreated mice (FIG. 11 (C)).
Immunostaining intensity is markedly increased in the L-NAME
salt-treated animals relative to both other groups. Glomerular
immunostaining intensity for CD31 did not vary between the
treatment, but confirm that the endothelin-limmunostaining is
primarily in the endothelial cell compartment. Collectively, the
data shows that endothelin expression is elevated with blood
pressure in Alport glomerular endothelial cells. This was not
observed in wild type glomeruli.
[0175] It is known that matrix metalloproteinases (MMPs),
transforming growth factor beta 1 (TGF-.beta.1), and monocyte
chemoattractant protein-1 (MCP-1) contribute to the progression of
Alport glomerular pathology (Cosgrove et al., 2000, Am J Pathol;
157(5):1649-59; Rao et al., 2006, Am J Pathol; 169(1):32-46;
Eisberg et al., 2006, PLoS Med; 3(4):e100). 129 Sv autosomal Alport
mice and wild type littermates were treated with 100 mg/kg Bosentan
or with carboxymethlycellulose vehicle by oral gavage from 2 to 7
weeks of age. One kidney was prepared for immunohistochemistry, and
the other used for glomerular RNA isolation. Real time RT-PCR
analysis of glomerular RNA (FIG. 12) showed a significant reduction
in the mRNA expression of MMP-10, MMP-12, TGF-.beta.1, and IL-6 in
glomeruli from Bosentan-treated Alport mice compared to Alport mice
given vehicle. MMP-9 expression was not affected; however this MMP
has been shown not to contribute to progression of Alport
glomerular disease (Andrews et al., 2000, Am J Pathol;
157(1):303-11).
[0176] Kidney cryosections from these same mice were immunostained
using antibodies against fibronectin, to assess interstitial
fibrosis, and CD11b, to assess the degree of monocytic interstitial
infiltration. FIG. 13 shows that kidneys from vehicle-treated
Alport mice showed significant renal scarring and massive monocytic
infiltration, consistent with what is normally observed in this
mouse model at 7 weeks of age. Bosentan treated mice showed near
complete blockade of both interstitial fibrosis and monocytic
infiltration, indicating a profound effect of endothelin receptor
blockade on Alport renal disease.
[0177] Given the effect of endothelin activation on Rac1 and CDC42
activation, and given the showings of Example 1, showing mesangial
process invasion of the glomerular capillaries in Alport mice (see
also Zallocchi et al., 2013, Am J Pathol; 183(4):1269-80), one
would expect that blockade would prevent or reduce mesangial
process invasion of the glomerular capillaries in treated mice.
FIG. 14 shows that for Alport mice given vehicle only, there is
extensive mesangial invasion of the glomerular capillaries, as
evidenced by the presence of integrin .alpha.8 immunostaining (a
mesangial cell surface marker) in regions immunopositive for
laminin .alpha.5 (a marker for the GBM). In Bosentan-treated Alport
mice, integrin .alpha.8 immunostaining is largely localized to the
mesangial matrix, with occasional interposition into the capillary
loops observed (denoted by arrowhead).
[0178] Collectively, FIGS. 11-14 demonstrate that endothelin-1 is
induced in Alport glomeruli by hypertension, and that endothelin
receptor blockade with Bosentan reduces glomerular expression of
MMPs and cytokines known to drive the progression of the disease,
ameliorates fibrosis and interstitial monocytic infiltration, and
blocks mesangial process invasion into the glomerular capillary
loops. This pathway (endothelin blockade on Alport glomerular
mesangial cells) represents a new etiology and thus a new use for
the drug as a treatment for Alport glomerular disease.
Example 3
Endothelin Receptor A and Integrin .alpha.8 Co-Localization
[0179] Using methods described in the examples included herewith,
FIG. 15 shows that the endothelin receptor A co-localizes with
integrin .alpha.8, which shows it is abundantly expressed on
mesangial cells in mice. While this has been previously shown for
rats, this example verifies the observation in mice, since our
model is for paracrine activation of mesangial ETRA by endothelin-1
which is of endothelial cell origin.
Example 4
Laminin 211-Mediated Focal Adhesion Kinase Activation Triggers
Alport Glomerular Pathogenesis
[0180] It has been known for some time that laminin 211 and 111,
normally restricted to the mesangial matrix, accumulate in the
glomerular basement membranes (GBM) of Alport mice, dogs, and
humans. This example shows that Laminin 211, but not laminin 111,
activates focal adhesion kinase (FAK) on glomerular podocytes in
vitro and in vivo. CD151-null mice also show progressive
accumulation of laminin 211 in the GBM, and podocyte FAK
activation. Analysis of glomerular mRNA from both models
demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs
linked to GBM destruction in Alport disease models, as well as the
pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured
podocytes significantly reduced expression of MMP-9, MMP-10 and
IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small
molecule inhibitor of FAK activation, ameliorated fibrosis and
glomerulosclerosis, significantly reduced proteinuria and blood
urea nitrogen levels, and partially restored GBM ultrastructure.
Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was
significantly reduced in TAE226 treated animals. Collectively, this
work identifies laminin 211-mediated FAK activation in podocytes as
an important early event in Alport glomerular pathogenesis and
suggests that FAK inhibitors might be employed as a novel
therapeutic approach for treating Alport renal disease in its early
stages.
[0181] The pathologic glomerular basement membrane in Alport
syndrome is irregularly thickened and thinned, with a multilaminar
or "basketweave" appearance that is unique to the disease and a
definitive diagnostic test for Alport syndrome (Kruegel et al.,
2013, Nat Rev Nephrol: 9: 170-178). It has been shown that the
thickened regions are more permeable to injected ferritin than the
non-thickened regions of the GBM (Abrahamson et al., 2007, J Am Soc
Nephrol: 18: 2465-2472). This property is consistent with a
partially degraded matrix network, suggesting proteolytic damage
may contribute to focal thickening of the Alport GBM. The type IV
collagen network in Alport GBM is comprised entirely of
.alpha.1(IV)/.alpha.2(IV) chains, which contains fewer interchain
crosslinks than the subepithelial
.alpha.3(IV)/.alpha.4(IV)/.alpha.5(IV) network found in wild type
GBM (Gunwar et al., 1998, J Biol Chem; 273: 8767-8775), and is more
susceptible to proteolytic degradation by endogenously expressed
matrix metalloproteinases (Rao et al., 2006, Am J Pathol; 169:
32-46; Zeisberg, et al., 2006, PLoS Med; 3: e1004.
[0182] Work on Alport renal disease thus far has focused on events
that occur after glomerular disease is well established. The work
includes roles for pro-inflammatory cytokines, such as TGF-.beta.1
(Zeisberg, et al., 2006, PLoS Med; 3: e100), CTGF (Koepke et al.,
2007, Nephrol Dial Transplant; 22: 1062-1069), and the mesangial
adhesion molecule all 1 integrin (Cosgrove et al., 2000, Am J
Pathol; 157: 1649-1659; Cosgrove et al., 2008, Am J Pathol; 172:
761-773), all of which contribute to the glomerular pathology in
Alport syndrome. MMPs are also induced as a function of disease
progression, and several MMPs, including MMP-2, MMP-9, and MMP-12
have been functionally linked to progressive destruction of the GBM
(Rao et al., 2006, Am J Pathol; 169: 32-46; Zeisberg, et al., 2006,
PLoS Med; 3: e100).
[0183] An unusual characteristic of Alport glomerular disease
progression is the early and progressive deposition of abnormal
laminins (laminin 211 and 111) in the GBM. While this phenomenon
was first described many years ago (Cosgrove et al., 2000, Am J
Pathol; 157: 1649-1659; Kashtan et al., 2001, J Am Soc Nephrol; 12:
252-260), the functional significance of this observation as it
relates to molecular pathology in the glomerulus has remained
unclear. As shown in Example, 1 laminin 211 in the GBM is deposited
by invading mesangial cell processes, a process that may be
triggered by biomechanical strain on the capillary tuft owing to
the altered type IV collagen composition of the GBM. In this
example work we identify FAK activation in podocyte foot processes
specifically in regions of the GBM where abnormal laminin
deposition is occurring. This is observed as early as P10, long
before detectable proteinuria for Alport mice on the 129 Sv/J
background, which is detectable at about 3 weeks of age (Cosgrove
et al., 2000, Am J Pathol; 157: 1649-1659). We link FAK activation
to elevated expression of MMP-9, MMP-10, MMP-12, and IL-6, all of
which are implicated in the progressive GBM destruction associated
with Alport glomerular disease. We demonstrate all of these
phenomena are also observed in the CD151 knockout mouse, which has
a specific defect in .alpha.3.beta.1 integrin binding affinity, a
characteristic likely to impact the structural integrity of the
capillary tuft as well (Zeng et al., 2006, Cancer Res; 66:
8091-8099). Importantly, the CD151 knockout mouse has a normal type
IV collagen network in the GBM, which suggests that these events
are not due to altered signaling resulting from the altered type IV
collagen basement membrane composition in Alport GBM.
Materials and Methods
[0184] Animals. Alport mice were either autosomal recessive (COL4A3
mutant on the 129 Sv/J background (Cosgrove et al., 1996, Genes
Dev; 10: 2981-2992). CD151 knockout mice were on the FVB background
and were a gift from Martin Hemler, Harvard Medical School (Takeda
et al., 2007, Blood; 109: 1524-1532). Laminin dy/dy mice were
obtained from the Jackson Laboratories (strain
#129P1/ReJ-Lama2dy/J, stock #000641). Age/strain matched wild type
mice were used as controls. All animal work was done under an IACUC
protocol approved by the BTNRH IACUC committee and in accordance
with the USDA and NIH guidelines for the care and use of animals
for research. Every effort was made to minimize stress and
discomfort.
[0185] Antibodies and inhibitors. Anti-.alpha.-actinin-4 was from
Santa Cruz Biotechnology, Inc (Dallas, Tex., USA, Cat #: SC-49333);
anti-CD11b was from CedarLane Laboratories Limited (Honrby,
Ontario, Canada, Cat #: CL8941AP); anti-Fibronectin was from Sigma
(St. Louis, Mo., USA, Cat #: F3648); anti-Integrin .alpha.8 was
from R&D Systems (Minneapolis, Minn., USA, Cat #: AF4076);
anti-Laminin .alpha.1 was a gift from Dr. Dale Abrahamson (KU
Medical Center, Kansas City, Kans., rat monoclonal 8B3);
anti-Laminin .alpha.2 and anti-3 actin were from Sigma (St. Louis,
Mo., USA, Cat #: L0663); anti-Laminin .alpha.5 was a gift from Dr.
Jeff Miner (Washington University, St. Louis, Mo.);
anti-p-FAK.sup.397 was from Assay Biotechnology (Sunnyvale, Calif.,
USA, Cat #: A0925) and from Invitrogen (Carlslab, CA); anti-Total
FAK was from Cell Signaling Technology (Danvers, Mass., USA, Cat #:
3285). All Alexa-fluor conjugated secondary antibodies were from
Invitrogen (Carlsbad, Calif.), including donkey anti-rat 488,
donkey anti-rabbit 555, goat anti-rat 488, goat anti-rabbit 555,
donkey anti-rabbit 488, and donkey anti-goat 568. The small
molecular inhibitor for FAK activation, TAE226 was from Chem Scene
(Monmouth Junction, N.J., Cat #CS-0594); the peptide inhibitor for
NF-kappaB (SN-50) was from Calbiochem (now EMD Millipore,
Billerica, Mass., Cat #481480).
[0186] Immunofluorescence microscopy. Fresh frozen kidneys were
sectioned at 8-Gim and acetone fixed. Sections were incubated
overnight at 4.degree. C. in primary antibody solution. The dual
stain consisting of rat anti-mouse Laminin-.alpha.2 antibody
(Sigma-Aldrich, St. Louis, Mo.) at 1:200 and rabbit anti-mouse
phospho-FAK 397 antibody at 1:25 as well as the dual stain of goat
anti-mouse Integrin .alpha.8 antibody (R & D Systems,
Minneapolis, Minn.) at 1:1000 and rabbit anti-mouse Laminin-5
antibody, at 1:1000 were diluted in 0.3% PBST+5% FBS. Rabbit
anti-mouse Fibronectin antibody at 1:300 and rat anti-mouse CD11b
antibody at 1:100 were diluted in 7% Milk. Slides were rinsed with
1.times.PBS and incubated with the appropriate Alexa Fluor donkey
secondary antibodies at 1:300 for 1 hour at room temperature. They
were then rinsed again with 1.times.PBS and mounted with
Vectashield Mounting Medium with DAPI (Vector, Burlingame, Calif.).
The dual stain of mouse-anti rat Laminin .alpha.1 antibody at 1:300
and rabbit anti-mouse phospho-FAK 397 antibody at 1:25 were diluted
in 0.3% PBST+5% NGS and incubated overnight at 4.degree. C. Slides
were rinsed with 1.times.PBS and incubated with the appropriate
Alexa Fluor goat secondary antibodies at 1:300 for 1 hour at room
temperature. They were then rinsed again with 1.times.PBS and
mounted with Vectashield Mounting Medium with DAPI.
[0187] Primary Mesangial Cells were derived and characterized as
previously described (Cosgrove et al., 2008, Am J Pathol; 172:
761-773). Three independent Transwell Migration Assays were
performed using 0.5 .mu.M TAE226 as previously described. For pFAK
Western Blot.sup.397, cells were maintained on 1% FCS-containing
media for two days, overnight in 0.1% BSA (Fraction V, Roche
Diagnostics, Mannheim, Germany) and TAE226 added to 0.5 and 1.0
.mu.M. After five hours protein was collected in M-PER.TM. (Thermo
Scientific, Rockford, Ill.) containing Protease Inhibitor Cocktail
P8340 at 1:100 (Sigma, St. Louis, Mo.), 5 mM Sodium Fluoride
(Sigma), and 2 mM Sodium Orthovanadate (Sigma) and Western Blots
run as described below.
[0188] Conditional Immortalized Glomerular Epithelial Cells
(GEC's), previously derived and characterized (Rao et al., 2006, Am
J Pathol; 169: 32-46), were grown under permissive conditions (10%
FCS, 10 U/ml .gamma.-interferon at 33.degree. C.). Stable FAK and
Scrambled Knock-Down GEC's were established as follows: 8.5 million
cells were electroporated in 0.5 mls Gene PULSER.TM.
Electroporation buffer (Bio-Rad Laboratories, Hercules, Calif.)
containing 20 .mu.g SILENCER.TM. 4.1 CMV neo (Ambion, Austin, Tex.)
plasmid expressing Ptk2 or scrambled siRNA, at 0.220 kV, 1.00
(.mu.F.times.1000) in a 4 mm gap cuvette and incubated for 10
minutes on ice. Cells were plated under permissive conditions and 2
mg/ml G418 (Invitrogen, Carlsbad, Calif.) was added three days
later. G418 selection was maintained for two weeks and clonal
populations of selected cells generated by "limiting-dilution." RNA
and protein was collected from expanded clonal populations placed
under "non-permissive" conditions (5% FCS, no .gamma.-interferon at
37.degree. C.) for two weeks, using TRIZOL.TM. (Invitrogen) and
M-PER.TM. (Thermo Scientific), respectively. Plasmid(s) Expressing
siRNA's were constructed using Ambion Silencer.TM.4.1-CMV neo,
AMBION SILENCER.TM. Select siRNA Ptk2 (ID s65838) and Negative
Control #1 (cat#AM4611) sequence(s) as per manufacturer's
direction.
[0189] NF-.kappa.B Staining and -/+Stretch pFAK397 Western Blot.
GEC's were differentiated under "non-permissive" conditions for ten
days, plated onto Bioflex 6-well plates (Flexcell International,
Hillsborough, N.C.) coated with Collagen Type 1 (rat tail, BD
Biosciences, Bedford, Mass.)/Placental Laminin (Sigma), cultured
for two days in 0.5% FCS and exposed to mechanical strain for 4
hours, as previously described (Meehan et al., 2009, Kidney Int;
76: 968-976). For NF-.kappa.B Staining, cells were fixed with 2%
PFA, 4% Sucrose in PBS for 10 minutes, permeabilized with 0.3%
Triton, as previously described (Rao et al., 2006, Am J Pathol;
169: 32-46) incubated with .alpha.NF-.kappa.B P65 antibody at 1:50
overnight at 4.degree. C., incubated with anti-rabbit secondary
antibody at 1:750 for two hours at room temperature, gaskets were
cut out, mounted on slides with VECTASHIELD.TM. (Vector
Laboratories, Burlingame, Calif.) and cover slipped. For -/+Stretch
pFAK Western, protein was collected in M-PER (Thermo Scientific) as
above and Western Blot run as described below.
[0190] NF-.kappa.B SN50, Inhibitor Peptide Treatment, GEC's were
cultured as described above, 10 .mu.M NF-.kappa.B SN50 Inhibitor
Peptide (EMD Millipore, Billerica, Mass.) was added (and after one
additional hour) exposed to 20 hours of mechanical strain and RNA
collected as previously described (Meehan et al., 2009, Kidney Int;
76: 968-976).
[0191] Basal Lamina and -/+TAE226 pFAK.sup.397 Western Blots. 10
day differentiated GEC's were cultured in 0.5% FCS for 2 days and
plated onto tissue culture dishes previously coated with 50
.mu.g/ml Collagen Type 1 Rat Tail (BD Biosciences) and 2
.mu.g/cm.sup.2 Placental Laminin (Sigma) in 0.1% BSA containing
media. For Basal Lamina experiment, additional dishes were coated
with Collagen Type 1 and 1.25 .mu.g/cm.sup.2 EHS Laminin (BD
Biosciences) or 1.25 .mu.g/cm.sup.2 Merosin (Chemicon, Temecula
Calif.). For -/+TAE266 experiment, 20 M TAE226 was included in the
media. Protein was collected 15 hours later in M-PER (Thermo
Scientific) as above and Western Blot run as described below.
[0192] Confocal microscopy. Slides were cover slipped using
Vectashield mounting medium containing DAPI to counter-stain the
nuclei (Vector, Burlingame, Calif.) and confocal images captured
using a Zeiss AxioPlan 2IF MOT microscope interfaced with a LSM510
META confocal imaging system, using a 63.times.NA: 1.4 oil
objective. Final figures were assembled using Adobe Photoshop and
Illustrator software (Adobe Systems, CA).
[0193] Glomerular isolation. Glomeruli were isolated by perfusing
animals with magnetic beads and isolating the glomeruli using a
magnet as described previously (Rao et al., 2006, Am J Pathol; 169:
32-46).
[0194] Real time qRT-PCR. Total RNA was reverse transcribed using
SUPERSCRIPT.RTM. III (Invitrogen, Life Technologies, Grand Island,
N.Y.) with Oligo(dT).sub.20 Primer (Invitrogen). The real time PCR
was carried out using TAQMAN.RTM. Gene Expression Master Mix
(Applied Biosystems, Life Technologies, Grand Island, N.Y.), and
quantified using STEPONEPLUS.TM. Real-Time PCR System (Applied
Biosystems). Samples were normalized to Mouse GAPDH Endogenous
Control VIC.RTM. Probe (Applied Biosystems catalogue #4352339E)
which was run alongside MMP-9 (Catalog #4331182, ID#Mm00442991_m1),
MMP-10 (Catalog #4331182, ID#Mm00444630_m1), MMP-12 (Catalog
#4331182, ID#Mm00500554_m1), IL-6 (Catalog #4331182,
ID#Mm00446190_m1), NFKbia (Catalog #4331182, ID#Mm00477798_m1), and
FAK (Catalog #4331182, ID#Mm00433209_m1) TAQMAN.RTM. Gene
Expression Assay Probes (Applied Biosystems). Samples were run in
triplicate with a final reaction volume of 20 ul with the following
cycling parameters: 50.degree. C. for 2 min, 95.degree. C. for 10
min, followed by 40 cycles of a two-step PCR consisting of
95.degree. C. for 15 s and 60.degree. C. for 1 min. Relative
changes in gene expression were determined by calculating the fold
change using the comparative C.sub.T method of
2.sup.-.DELTA..DELTA.CT Data are expressed as the mean with
standard deviation for at least four independent RNA samples per
data point.
[0195] Immunoblotting. Ten to fifteen micrograms, of cellular
protein, was resolved in a 10% SDS-PAGE and then electrotransfered
to PVDF membrane. The membranes were cut in half and the upper half
(250 kDa to 75 kDa) used for pFAK/tFAK immunoblotting while the
bottom half used for .beta.-actin immunoblotting (loading control).
Conditions for pFAK detection: the membrane was blocked in milk
blocking solution (5% milk containing 0.2% Tween-20 in PBS) for 1
hour at room temperature with constant shaking and incubated
overnight at 4.degree. C. with anti-pFAK 1:1,000 in BSA blocking
solution (1% BSA containing 0.2% Tween-20 in PBS). After several
washes the membrane was incubated with a goat anti-rabbit HRP
conjugated secondary antibody in BSA blocking solution, dilution
1:20,000 for 1 hour at room temperature. Conditions for tFAK
detection: the same membrane used for pFAK immunoblot was stripped
and re-probed for tFAK. The blocking was done in 5% milk blocking
solution for 1 hour at room temperature, followed by an overnight
incubation with the tFAK primary antibody, dilution 1:500 in milk
blocking solution. After several washes the membrane was incubated
with a goat anti-rabbit HRP conjugated secondary antibody in 5%
milk, dilution 1:3,000 for 1 hour at room temperature. Conditions
for .beta.-actin detection: the membrane was blocked for 1 hour
with 10% milk blocking solution and then incubated overnight with
the mouse anti-.beta.-actin dilution 1:2,000 in the same blocking
solution. After several washes the membrane was incubate with a
goat anti-mouse HRP-conjugated secondary antibody, dilution 1:3,000
in 10% milk for 1 hour at room temperature. After several washes
the membrane was developed using PIERCE.RTM. ECL Western Blotting
Substrate (Thermo Scientific, Rockford, Ill.) as per manufacturer's
direction.
[0196] Treatment of mice with TAE226. Four Col 4A3 knockout mice
from 129 Sv/J background were given 50 mg/Kg TAE226 (ChemScene, LLC
Monmouth Junction, N.J.) 1.times. daily by gavage needle starting
at two weeks of age until seven weeks old. The TAE226 was diluted
in a 0.5% carboxy methyl cellulose suspension. Three control Col
4a3 knockout mice of the same age were given 0.5% CMC suspension
alone and served as controls.
[0197] Albumin and creatinine assays. Urine was collected weekly
and albumin concentrations were analyzed as instructed using a
mouse albumin ELISA kit #MSAKT from Molecular Innovations (Novi,
Mich.). Albumin levels were normalized to creatinine using
QuantiChrom Creatinine Assay Kit (DICT-500) (BioAssay Systems,
Hayward, Calif.) as instructed.
[0198] Transmission electron microscopy. Transmission electron
microscopy was performed as described previously (Cosgrove et al.,
2000, Am J Pathol; 157: 1649-1659).
[0199] Statistical analysis. Data were analyzed using the one
sample Students t-test with Bonferroni correction.
Results
[0200] In earlier work we and others showed that laminin 211
accumulates in the GBM of Alport mice, dogs and humans (Cosgrove et
al., 2000, Am J Pathol; 157: 1649-1659; Kashtan et al., 2001, J Am
Soc Nephrol; 12: 252-260). FIG. 16 shows that the appearance of
laminin 211 in the GBM correlates with the activation of FAK in
glomerular podocytes. FIG. 16 (A-C) shows that in wild type mice
laminin 211 is restricted to the mesangium and no appreciable level
of FAK activation (as determined by immunostaining for
pFAK.sup.397) is observed. As early as 10 days of age in 129 Sv/J
autosomal Alport mice we begin to observe punctate immunostaining
for laminin 211 in the GBM of some (30-50%) glomeruli (FIG. 16 (D),
arrowheads). Wherever we observe GBM laminin staining we also see
immunopositivity for pFAK.sup.397 (FIG. 16 (E-F)), indicating
activation of FAK specifically in regions of the GBM where laminin
211 has been deposited. By 7 weeks of age in Alport glomeruli,
laminin 211 is more extensively observed in the GBM (FIG. 16 (G)),
and continues to co-localize with pFAK.sup.397 immunostaining (FIG.
16 (G-I)).
[0201] In addition to laminin 211, laminin 111 has also been shown
to accumulate in the GBM of Alport mice (Abrahamson et al., 2003,
Kidney Int; 63: 826-34). To determine whether laminin 211 and/or
laminin 111 is responsible for activation of FAK in glomerular
podocytes we crossed the 129 Sv/J autosomal Alport mouse with a
laminin .alpha.2-deficient mouse (a model for muscular dystrophy),
also on the 129 Sv/J background. As evidenced in FIG. 17, while the
7 week old Alport mouse shows FAK activation in podocytes bound to
laminin 111-immunopositive GBM (FIG. 17 (A-C)), the age matched
laminin .alpha.2-deficient Alport mouse (DY Alport), while
immune-positive for laminin 111 (FIG. 17 (D)), does not show
appreciable FAK activation anywhere in the glomerulus (FIG. 17
(E)). To assess in a more direct manner whether laminin 211
activates FAK in podocytes, we cultured differentiated
conditionally immortalized podocytes on placental laminin
(primarily laminin 521), EHS laminin (laminin 111), and merosin
(laminin 211) for 15 hours and analyzed cell lysates for total FAK
and pFAK.sup.397. The results in FIG. 17 (G) show higher levels of
pFAK.sup.397 in podocytes cultured on merosin compared to either
placental laminin or EHS laminin, indicating that laminin 211 can
activate FAK directly in these cells.
[0202] As shown in Example 1, laminin 211 also accumulates in the
GBM of CD151 knockout mice (see also Zallocchi et al., 2013, Am J
Pathol; 183: 1269-80). If laminin 211 is responsible for FAK
activation on glomerular podocytes in vivo we would expect to
observe pFAK.sup.397 immunostaining at the interface of podocyte
binding to the GBM in these mice as well. FIG. 18 shows that this
is indeed the case. FIG. 18 (D-F) clearly demonstrates laminin 211
immunostaining in the GBM with clear presence of pFAK.sup.397 in
podocytes adjacent to laminin 211-immunopositive GBM, consistent
with laminin 211 mediated FAK activation in the podocytes of CD151
knockout mice.
[0203] A clear link between the induction of matrix
metalloproteinases and glomerular basement membrane damage has been
demonstrated in Alport mice (Rao et al., 2006, Am J Pathol 169:
32-46; Zeisberg et al., 2006, PLoS Med 3: e100; 9; and Cosgrove et
al., 2008, Am J Pathol 172: 761-773). Based on Affymetrix analysis
of wild type and Alport glomerular RNA from 129 Sv/J mice, it was
determined that MMP-9, MMP-10, and MMP-12 were significantly
induced in the Alport glomeruli. MMP-10 and 12 are massively
induced (700- and 40-fold, respectively), suggesting that these
MMPs might be principally responsible for the GBM damage observed
in Alport mice. Given that previous studies in other systems have
linked FAK activation to the induction of MMPs (Zeng et al., 2006,
Cancer Res; 66: 8091-8099; Van Slambrouch et al., 2007, Int J
Oncol; 31:1501-1508), it was determined if a parallel dysregulation
in glomerular RNA from Alport mice and CD151 knockout mice could be
observed. Glomerular mRNA expression was profiled for a time course
in both models using real time qRT-PCR. The results in FIG. 19 (A)
demonstrate significant and progressive induction of all three MMPs
in both models. The strikingly robust induction of MMP-10 and
MMP-12 observed in Alport glomeruli is also observed in the CD151
knockout mouse, suggesting that these transcripts are induced via
the laminin 211-mediated FAK activation pathway. Since earlier work
demonstrates FAK-mediated induction of MMPs via activation of
NF-kappaB (Chen et al., 2009, J Cell Physiol; 221: 98-108; Oh et
al., 2009, Gynecol Oncol; 114: 509-515), NF-kappaBia transcript,
which serves as an indicator for the state of NF-kappaB activation
(Bottero et al., 2003, Mol Diagn; 7: 187-194), was also observed.
As shown in FIG. 19 (A), this transcript trends higher in
glomerular RNA for both models, as does the message encoding the
NFkappaB-responsive pro-inflammatory cytokine IL-6 (Tseng et al.,
2010, J Cell Physiol; 223: 389-396). Neither transcript shows
significant induction due to a high degree of variability in
abundance, likely owing to multiple pathways (in addition to FAK)
converging on the activation of NF-kappaB.
[0204] MMP-10 expression in the glomerulus has not been previously
documented. To further qualify the validity of the qPCR results, we
analyzed cryosections of 4 and 7 week old wild type and Alport mice
for MMP-10 expression by immunofluorescence. The results in FIG. 19
(B) show that MMP-10 is not detected in wild type glomeruli, but is
robustly expressed in Alport glomeruli at both early and advanced
disease states.
[0205] To more directly establish the link between FAK activation
and MMP gene expression in glomerular podocytes we performed siRNA
knockdown of FAK in conditionally immortalized podocyte cell
cultures. Stable clonal populations of siRNA knockdown podocyte
cell lines were established. FIG. 20 shows results typical for
several clones examined. In FIG. 20 (B), note the relative absence
of focal adhesions in podocytes cultured on rat tail collagen
relative to the parent podocyte cell line shown in FIG. 20 (A).
FIG. 20 (C) shows that total FAK protein is reduced in extracts
from the siRNA knockdown cells relative to cells transfected with a
scrambled siRNA construct. FIG. 20 (D) shows that FAK knockdown
cells show significantly reduced expression of MMP-9, MMP-10, and
NF-kappaBia, confirming the link between FAK activation and
induction of these MMPs in glomerular podocytes. Interestingly
MMP-12 was not significantly reduced in the knockdown cells.
[0206] An alternative means of reducing FAK activation is by way of
small molecule inhibitors. One such inhibitor, TAE226 has been
shown to protect against glomerular injury by either
lipopolysaccharide or anti-GBM antibody administration. Podocytes
were cultured in the presence or absence of TAE226 to assess the
effect on MMP expression. As shown in FIG. 21 (A) shows that
treatment of cultured podocytes with TAE226 reduced the activation
state of FAK and that FAK is directly activated by biomechanical
stretching of podocytes as determined by western blot for pFAK397
protein. FIG. 21 (B-C) shows that, in contrast to the siRNA
knockdown studies, both MMP-10 and MMP-12 show reduced expression
in podocytes cultured with TAE226 relative to untreated cells.
Since we have previously documented a role for biomechanical strain
in the induction of MMPs and the acceleration of glomerular disease
in Alport mice (Zallocchi et al., 2013, Am J Pathol; 183: 1269-80;
Meehan et al., 2009, Kidney Int; 76: 968-976) we also assessed the
effect of FAK inhibition by TAE226 on biomechanical
stretch-mediated induction of MMP-10 and MMP-12. FIG. 21 (B-C)
shows, consistent with our earlier findings, that biomechanical
stretch induced both MMP-10 and MMP-12, and that message levels for
these two MMPs are reduced in cells stretched in the presence of
TAE226 relative to untreated cells.
[0207] FIG. 22 shows that biomechanical stretching activates
NF-kappaB as evidenced by the nuclear localization of NF-kappaB in
FIG. 22 (B) relative to non-stretched cells shown in FIG. 22 (A).
Stretch-mediated induction of MMP-10 is attenuated by treating
cells with a peptide inhibitor for NF-kappaB during the cyclic
mechanical stretching (FIG. 22 (C)), demonstrating that MMP-10
induction is indeed mediated by NF-kappaB activation.
[0208] To determine the role of laminin 211-mediated FAK activation
on the progression of glomerular disease autosomal Alport mice were
treated with TAE226 from 2 weeks of age (before the onset of
proteinuria) to 7 weeks of age (near end stage). One kidney was
used for glomerular RNA isolation by perfusion with magnetic beads,
and the other prepared for histological and TEM analysis. FIG. 23
shows FAK activation in podocytes adjacent to laminin 211 in the
Alport GBM (denoted with arrowheads). Treatment with TAE226
abolished pFAK immunostaining (D and F) demonstrating effective in
vivo blockade of FAK activation achieved through drug treatment.
FIG. 23 (G) shows that FAK inhibition significantly reduced the
mRNA expression levels for MMP-9, MMP-10, and MMP-12 relative
Alport mice given vehicle. FIG. 23 (H-I) show a significant
reduction in proteinuria and blood urea nitrogen levels in treated
Alport mice relative to Alport mice given vehicle at 6 weeks of
age, but the numbers, while trending lower, loose significance by 7
weeks of age. Lifespan studies were not conducted because TAE226
treatment resulted in growth stunting indicating a toxic side
effect.
[0209] As shown in Example 1, progressive mesangial invasion of the
glomerular capillary loops has been shown in Alport mice (see also
Zallocchi et al., 2013, Am J Pathol; 183: 1269-80). FIG. 24 (A-F)
shows dual immunofluorescence staining for the GBM marker laminin
.alpha.5 and the mesangial cell surface marker integrin .alpha.8.
Mesangial processes in the capillary loops are clearly observed in
the vehicle treated Alport glomeruli (FIG. 24 (C), arrowheads,
inset panel). TAE226 treatment resulted in amelioration of
mesangial process invasion (FIG. 24 (E-F), inset panels; showing
integrin .alpha.8 immunostaining only at the mesangial angles),
suggesting that FAK activation on mesangial cells may contribute to
this process mechanistically. Consistent with this notion,
treatment of mesangial cells with TAE226 significantly reduced
their cell migratory potential (FIG. 24 (K)) and blocked pFAK
activation in a dose-dependent manner (FIG. 24 (L)). Transmission
electron microscopic analysis of the GBM in TAE226-treated animals
showed markedly improved GBM architecture relative to mice given
vehicle (FIG. 24 (G-J)).
[0210] To evaluate the effect of TAE226 treatment on renal
fibrosis, kidney sections were stained with antibodies specific for
either fibronectin (to assess renal scarring) or CD11b (to assess
for monocytic infiltration). The results in FIG. 25 show that
TAE226 treatment results in remarkably robust reduction in both
renal scarring (A-C) and monocytic infiltration (D-F). In all four
treated mice it was difficult to distinguish immunostaining of wild
type kidneys from TAE226 treated Alport kidneys using these two
antibodies.
Discussion
[0211] The initially punctate and then progressive deposition of
laminin 211 and laminin 111 in the GBM of Alport mice is a
phenomenon that would be expected to have some consequence
contributing to the progressive deterioration of the glomerular
structure/function, although until now no definitive functional
consequence has been described. This example provides evidence that
laminin 211 activates FAK on glomerular podocytes resulting in
downstream activation of MMPs and pro-inflammatory cytokines that
contribute to the progressive glomerular pathogenesis. At least
some of these genes are induced by NF-kappaB activation, suggesting
that the laminin 211/FAK/NF-kappaB circuit might be a central
player driving the progression of Alport glomerular disease. In
support of this notion, treatment of Alport mice with a small
molecule inhibitor for FAK, TAE226, resulted in a significant
reduction in glomerular expression of MMP-9, MMP-10, and MMP-12,
improved glomerular function, ameliorated ultrastructural damage to
the GBM, and blocked interstitial monocyte infiltration and
interstitial fibrosis. In spite of what appears to be significantly
improved ultrastructure, proteinuria in the TAE226 treated Alport
mice, while lower than vehicle-treated mice, was still relatively
high. This may be due to proteolytically induced microlesions in
the GBM caused by troughs in the inhibitory activity of TAE226
(pharmacokinetics), or more likely due to pathways other than FAK
that contribute to the glomerular pathogenesis.
[0212] While the effects of TAE226 on FAK activation in glomerular
podocytes is likely the principal contributing factor underlying
the observed improvement of the GBM ultrastructure and function, it
is also likely that the systemic administration of this compound
might have multiple influences on improved renal health in these
animals. For example, as shown in Example 1, laminin 211 is
deposited in the GBM by mesangial processes that invade the
glomerular capillaries (see also Zallocchi et al., 2013, Am J
Pathol; 183: 1269-80). This example shows that TAE226 treatment
reduced the degree of mesangial process invasion (FIG. 24 (D-F))
and reduces the migratory potential of cultured mesangial cells
(FIG. 24 (J)), suggesting that mesangial processes invasion of
glomerular capillaries in Alport syndrome might be partially
FAK-dependent. This makes some sense considering that deletion of
integrin .alpha.1.beta.1, a major mesangial cell surface integrin,
also ameliorates mesangial process invasion of the glomerular
capillaries in Alport mice, and significantly improves renal health
in this model (Cosgrove et al., 2000, Am J Pathol; 157: 1649-1659;
Zallocchi et al., 2013, Am J Pathol; 183: 1269-80). The remarkable
reduction in interstitial monocytes might reflect a third distinct
activity for FAK in Alport renal disease. In earlier work we showed
that interstitial monocytes in a mouse model are primarily
.alpha.1.beta.1 integrin-positive (Sampson et al., 2001, J Biol
Chem; 276: 34182-34188). It was later shown that .alpha.1.beta.1
integrin-positive monocytes are selectively trafficked to the
interstitium n Alport kidneys (Dennis et al., 2010, Am J Pathol;
177: 2527-2540). In other systems it has been shown that leukocyte
activation following tight binding to the vascular endothelium can
be mediated through FAK signaling (Li et al., 1998, J Biol Chem;
273: 9361-9364). Thus inhibiting monocyte activation to reduce
interstitial monocyte efflux might represent a third target of FAK
inhibitors that improves renal health in Alport mice.
[0213] This example demonstrates using both in vitro (FIG. 17 (G))
and in vivo (FIG. 17 (A-F)) approaches that laminin 211, but not
laminin 111, activates FAK on glomerular podocytes. This is an
important distinction when one considers that abnormal laminins
have been shown to accumulate in the GBM in patients with
membranous glomerulonephritis (Horikoshi et al., 1999, Nephron; 81:
284-288; and Fischer et al., 2000, Nephrol Dial Transplant; 15:
1959-1994) where much like the Alport model, the laminins are first
observed in the irregularly thickened regions of the GBM. In the
Alport mouse model, irregularly thickened abnormal laminin-rich
regions of the GBM were shown to be more permeable to injected
ferritin, suggesting that these regions are comprised of loosely
assembled matrix that might contribute to progressive leakiness and
proteinuria (Abrahamson et al., 2007, J Am Soc Nephrol 18:
2465-2472.). Based on our observation of FAK mediated induction of
MMP-9, MMP-10, and MMP-12 in the Alport podocyte, it appears that
the increased GBM permeability in these thickened regions might
reflect partially degraded GBM. Earlier studies found that deletion
of MMP-9 in Alport mice did not influence renal disease
progression, suggesting that MMP-9 may not contribute significantly
to the pathology (Andrews et al., 2000, Am J Pathol; 157:
303-311).
[0214] The accumulation of abnormal laminins in the GBM may be more
generally applicable to glomerulonephritis. It will be important to
determine specifically which abnormal laminin heterotrimers
accumulate in the GBM in membranous glomerulonephritis where GBM
deposition of laminin .beta.1 has been described, whether FAK is
activated, and whether elevated glomerular expression of MMPs is
observed. Such data would implicate the use of FAK blockade as a
potential therapeutic approach for this glomerular disease as well
as for Alport syndrome. A recent study using both
lipopolysaccharide (LPS) and anti-GBM antibody-induced glomerular
disease models showed that podocyte injury could be limited by
blocking FAK activation (Ma et al., 2010, J Am Soc Nephrol; 21:
1145-1156), providing further evidence of the general utility of
FAK inhibitors for the treatment of glomerular diseases.
[0215] Laminin 211-mediated FAK activation is also observed in the
CD151 knockout mouse model (FIG. 18). Like the Alport mouse, the
CD151 knockout mouse shows massive up-regulation of both MMP-10 and
MMP-12 (FIG. 19), providing further evidence that induction of
these genes is regulated by FAK activation. MMP-10 expression in
the glomerulus has not been previously documented, likely owing to
its low abundance in healthy glomeruli. Immunostaining for MMP-10
(FIG. 19 (B)) showed that MMP-10 is undetectable in the wild type
glomeruli and abundant in the Alport glomeruli. MMP-10, like its
related stromelysin MMP-3, has a broad substrate specificity, which
includes type IV collagen (Sanchez-Lopez et al., 1993, J Biol Chem;
268: 7238-7247; and Nagase H., 2001, Substrate Specificity of MMPs.
Cancer Drug Discovery and Development: Matrix Metalloproteinase
Inhibitors in Cancer Therapy, edited by Clendeninn J J and
Krzysztof A. Totowa, N.J., Humana Press Inc., 39-66). The high
levels of induction observed (700- to 1200-fold) suggest that
MMP-10 might play an important role in the pathogenic mechanism of
Alport glomerular disease, warranting further study.
[0216] As shown in Example 1, biomechanical strain, most likely
owing to the change in basement membrane composition, has
pro-pathogenic consequences in Alport glomeruli. These include
exacerbating GBM destruction by way of MMP induction and
accelerating the invasion of glomerular capillaries by mesangial
processes (see also Zallocchi et al., 2013, Am J Pathol; 183:
1269-80; Meehan et al., 2009, Kidney Int; 76: 968-976). This
example shows that biomechanical stretching of podocytes directly
activates FAK and induces the expression of MMP-10 and MMP-12 (FIG.
21). Treatment of stretched cells with the FAK inhibitor TAE226
blocked MMP induction in this same set of experiments. This example
also showed that stretching podocytes caused nuclear localization
of NF-kappaB (FIG. 22), consistent with its activation (Beg et al.,
1992, Genes Dev; 6: 1899-1913), and that adding a peptide inhibitor
for NF-kappaB to stretched cells blocked the induction of MMP-10.
Collectively, these data suggest that biomechanical strain
exacerbates laminin 211-mediated activation of FAK in podocytes
leading to NF-kappaB-dependent induction of MMP-10.
[0217] This example defines a role for GBM laminin 211 in Alport
glomerular pathogenesis by way of activation of FAK on glomerular
podocytes leading to the downstream activation of MMP-9, MMP-10,
and MMP-12 gene expression. This mechanism of MMP induction
involves NF-kappaB activation and is exacerbated by biomechanical
strain on the glomerular capillary tuft. Further, this example
shows that systemic inhibition of FAK by way of a small molecule
inhibitor ameliorates both glomerular and tubulointerstitial
pathologies, likely owing to its effects not only on glomerular
podocytes, but also mesangial cells, and possibly firm
adhesion-mediated monocyte activation. Since laminin 211 starts to
be deposited in the Alport GBM as early as 10 days in the 129 Sv/J
autosomal mouse model, where proteinuria is not detected until 3
weeks of age, we propose that this represents one of the earliest
events underlying the development of Alport glomerular disease.
This mechanism may be generally applicable to other forms of
glomerulonephritis where the accumulation of "abnormal" laminins in
the GBM has been documented.
[0218] This example has now published as "Laminin a2-mediated focal
adhesion kinase activation triggers Alport glomerular
pathogenesis," Delimont D, Dufek B M, Meehan D T, Zallocchi M,
Gratton M A, Phillips G, Cosgrove D, PLoS One, 2014 Jun. 10;
9(6):e99083, doi: 10.1371/joumal.pone.0099083, eCollection 2014,
which is hereby incorporated by reference in its entirety.
Example 5
Endothelin A Receptor Blockade Prevents Mesangial Filopodial
Invasion of Glomerular Capillaries and Delays Alport Glomerular and
Interstitial Disease Onset
[0219] The type IV collagen network in Alport glomerular basement
membrane (GBM) is comprised of only .alpha.1(IV) and .alpha.2(IV)
chains which contain fewer interchain crosslinks than the
.alpha.3(IV)/.alpha.4(IV)/.alpha.5(IV) networks found in normal
GBM. The presumed resulting increase in elasticity of the Alport
GBM imparts biomechanical stresses on the cell contacts comprising
the capillary tuft, which activates the Rho GTPases Rac1 and CDC42
in mesangial cells, inducing the invasion of the capillary tufts by
mesangial filopodia. The filopodia deposit mesangial proteins in
the GBM, which activate aberrant proinflammatory cell signaling in
podocytes. Thus, CDC42 activation is the molecular trigger for
Alport glomerular disease initiation.
[0220] With this example, 129 autosomal Alport mice were given
either Bosentan (an endothelin A and B receptor antagonist) or
Sitaxentan (an endothelin A receptor antagonist) from 2 to 7 weeks
of age. Mice were analyzed longitudinally for proteinuria and BUN,
glomerular RNA for gene expression of MMPs and pro-inflammatory
cytokines by real time RT-PCR, and tissue was analyzed
histochemistry and immunohistochemistry for pathologic changes.
[0221] Hypertension elevated expression of endothelin-1 in Alport
endothelial cells, but not wild type endothelial cells. Endothelin
blockade in Alport mice significantly reduced the mesangial
filopodial invasion of glomerular capillaries. This was associated
with delayed onset and slowed progression of proteinuria, and
increase in lifespan. GBM dysmorphology was ameliorated, and
glomerulosclerosis and interstitial fibrosis were not evident in
treated Alport mice when age-matched vehicle-treated Alport mice
showed >30% glomerulosclerosis and fibrosis scores between III
and IV. Both Bosentan and Sitaxentan were equally effective at
ameliorating Alport renal disease, likely because mesangial cells
were found to express only the endothelin A receptor.
[0222] In conclusion, biomechanical strain-mediated activation of
endothelin expression in Alport endothelial cells results in
endothelin A receptor-mediated activation of CDC42 in mesangial
cells, inducing the invasion of the subendothelial aspect of the
GBM by mesangial filopodia and is an important factor contributing
to the mechanism of Alport disease initiation, and presents a host
of novel therapeutic targets with the potential to delay/inhibit
the onset of Alport glomerular and tubulointerstitial
pathogenesis.
Example 6
Early Mechanisms of Alport Glomerular Pathology
[0223] The cellular origin of glomerular basement membrane (GBM)
laminin 211 has not been previously determined. As shown in Example
1, the source of GBM laminin 211 in Alport GBM is mesangial cell
processes, which are invading the capillary tufts. Salt-mediated
hypertension exacerbates this mesangial process invasion.
Deposition of laminin 211 in the GBM activates focal adhesion
kinase in podocytes, which leads to NF-kappaB activation and
induction of pro-inflammatory cytokines as well as MMPs, driving
the progression of Alport glomerular disease. A knockout mouse for
the integrin .alpha.3.beta.1 co-receptor CD151, which results in
reduced adhesion of podocytes pedicles to GBM laminin 521, also
develops mesangial process invasion of the capillary loops with GBM
deposition of laminin 211, demonstrating the same phenotype for a
completely unrelated molecular component of the glomerular
capillary structural barrier. See also, Zallocchi et al., 2013, Am
J Pathol; 183(4):1269-80.
[0224] The CD151 knockout mouse model also shows accelerated
glomerular disease progression in response to hypertension ((Sachs
et al., 2012, J Clin Invest; 122(1):348-58). As shown in the
previous examples, biomechanical stretching of cultured mesangial
cells induces pro-migratory cytokines TGF-.beta.1 and CTGF, both
known to be induced in Alport glomeruli (Sayers et al., 1999,
Kidney Int; 56(5):1662-1673; and Koepke et al., 2007, Nephrol Dial
Transplant; 22(4): 1062-9). Using inhibitor studies in Example 1,
it has been shown that mesangial cell migration is mediated by the
Rho GTPase RAC1 and that treatment of Alport mice with a RAC1
inhibitor blocks mesangial process invasion of the glomerular
capillary tufts, clearly implicating the activation of Rac1 in this
process (see also Zallocchi et al., 2013, Am J Pathol;
183(4):1269-80). These data define a surprising role for
biomechanical strain mediated-induction of mesangial cell process
invasion as a key aspect of Alport glomerular disease initiation,
and set the stage for defining novel therapeutic targets aimed at
blocking this process.
[0225] Below, it is also shows that endothelin 1 is induced in the
endothelial cells of Alport mice at an early age (before the onset
of proteinuria), and that expression is further induced by
hypertension. It has been previously shown that endothelin 1
activates CDC42/RAC1 in glomerular mesangial cells via activation
of endothelin receptors (Chahdi et al., 2005, J Biol Chem;
280(1):578-84; Chahdi and Sorokin, 2006, Exp Biol Med; 231(6):
761-5), and it is well established that biomechanical stretching
induces endothelin 1 expression and secretion by endothelial cells
(Just et al., 2004; Babu et al., 2012). It is also shown that ET-1
treatment of primary cultured mesangial cells activates CDC42 and
induces the formation of drebrin-positive actin microspikes (FIG.
30). Thus, it is likely that activation of RAC1/CDC42-mediated
actin cytoskeletal dynamics associated with mesangial process
invasion of the glomerular capillary tufts in Alport syndrome is
caused by endothelin-1 expression/secretion induced in the
glomerular endothelial cells by biomechanical strain.
[0226] While the presence of abnormal laminins in the Alport GBM
was described 14 years ago (Cosgrove et al., 2000, Am J Pathol;
157(5):1649-59; Kashtan et al., 2001, J Am Soc Nephrol; 12:252-60),
the functional significance of this observation as it relates to
molecular pathology in the glomerulus has, until now, remained
unknown. As shown in Example 4, FAK activation in podocyte foot
processes is identified specifically in regions of the GBM where
abnormal laminin deposition is occurring (see also Delimont et al.,
PLoS One, 2014 Jun. 10; 9(6)). This is observed as early as P10,
long before detectable proteinuria for Alport mice on the 129 Sv
background (about 3 weeks). We have determined the cellular source
of GBM laminin 211 to be mesangial filopodia. If the formation of
filopodia is blocked by way of a small molecule inhibitor for
RAC1/CDC42 Rho GTPases, laminin 211 deposition is largely blocked
in the Alport GBM (see Example 1 and Zallocchi et al., 2013, Am J
Pathol. 2013 October; 183(4):1269-80). As shown below, hypertension
results in markedly elevated endothelin-1 expression in the
glomerular endothelial cell compartment in pre-proteinuric Alport
mice, but not in wild type mice. When endothelin receptors in
Alport mice are blocked with small molecule inhibitors for both
endothelin A and B receptors (Bosentan) or one specific for
endothelin A receptors (Sitaxentan), mesangial process invasion of
the glomerular capillaries was markedly reduced, delaying the onset
and progression of proteinuria, ameliorating GBM structural
abnormalities, and significantly reducing glomerular expression of
MMPs and pro-inflammatory cytokines. Collectively, these studies
define a paradigm shift in our understanding of glomerular
pathology in Alport syndrome, and define a key mechanism of
glomerular disease initiation where biomechanical strain mediated
induction of endothelin-1 in glomerular endothelial cells activates
RAC1/CDC42 GTPases in mesangial cells. RAC1/CDC42 activates actin
cytoskeletal dynamics in mesangial cells, resulting in the invasion
of glomerular capillaries by mesangial filopodia. The filopodia
deposit laminin 211 in the GBM, which activates FAK in podocytes
resulting in elevated expression of pathologic genes, and revealing
a key molecular mechanism underlying podocyte dysfunction in Alport
syndrome. The discovery of this pathway reveals novel opportunities
for therapeutic intervention that were previously inconceivable,
including endothelin blockade using drugs already FDA approved for
the treatment of pulmonary hypertension. This example will
rigorously test this pathway by way of in vivo genetic modeling and
in vitro cell culture studies, aiming to define new endothelial
cell-specific therapeutic targets that can dislocate the
strain-dependent induction of endothelin-1.
[0227] The presence of abnormal laminins in the GBM, likely due to
reduced ILK activity, results in activation of FAK, which is a
critical step in disease initiation. The GBM laminin 211 is
deposited by mesangial filopodial processes that invade the GBM as
a result of biomechanical strain-mediated RAC1/CDC42 activation.
This is a new direction from conventional thinking regarding the
pathobiology of Alport glomerular disease. This is shown in Example
1 (see also Zallocchi et al., 2013, Am J Pathol; 183(4):1269-80),
and the extension of this work forms the foundation of this
example. The fact that endothelin A blockade blocks this process,
and has such a profound effect on Alport glomerular pathology
predicts that this mechanism is centrally important and provides an
opportunity for developing effective therapeutic approaches that
target this pathway.
[0228] FAK activation is directly linked to the induction of actin
cytoskeletal rearrangement (and thus contributing to foot process
effacement) and results in maladaptive gene regulation including
massive up-regulation of MMP-10 and MMP-12 in podocytes. This
turned out to be entirely true. FIGS. 26-31 are provided to show
the evidence for this mechanism. It turns out that laminin 211, but
not laminin 111, does activate FAK, and that FAK inhibition using
small molecule inhibitors ameliorate MMP-10 and MMP-12 induction,
GBM ultrastructural abnormalities, and renal fibrosis in Alport
mice. This work is shown in Example 4 (see also Delimont et al.,
PLoS One. 2014 Jun. 10; 9(6):e99083).
[0229] Massive induction of MMP-10 and MMP-12 in glomerular
podocytes causes proteolysis of the GBM resulting in proteinuria
and progression towards glomerulosclerosis, playing a major role in
the onset and the rate of progressive GBM pathogenesis in Alport
syndrome. Surprisingly, these two MMPs, which are massively induced
in Alport glomerular RNA, do indeed influence progression of
glomerular pathology, the effect of deleting them was rather mild.
Thus it is likely there are other proteases that play a dominant
role in the irregular thickening of the GBM and in the evolution of
proteinuria. The small molecules used in previous studies have
broad inhibitory effects ((Rao et al., 2006, Am J Pathol; 169:
32-46; Zeisberg, et al., 2006, PLoS Med; 3: e100), and predict that
these proteases are indeed important to glomerular pathology. The
specific proteases that dominate this influence remain to be
discovered. Nonetheless, work on MMP-10 and MMP-12 is important,
because it rules out a dominant role for these metalloproteinases
in Alport GBM destruction. The experiments of this example are
upstream of proteolytic degradation of the GBM, given that
endothelin A receptor blockade largely prevents irregular GBM
dysmorphology.
Research Design and Methods
[0230] It has been previously shown that hypertension accelerated
the progression of Alport glomerular disease, suggesting a key role
for biomechanical strain in the disease mechanism (Meehan et al.,
2009, Kidney Int; 76: 968-976). This the data demonstrating the
induction of mesangial process invasion of the glomerular
capillaries suggests that biomechanical stretching of the capillary
tuft might activate actin cytoskeletal dynamics in Alport
glomeruli. As shown in Example 1, this observation was extended
with the discovery that mesangial processes invade the glomerular
capillaries in a biomechanical strain-mediated
Rac1/CDC42-activation mechanism (see also Zallocchi et al., 2013,
Am J Pathol; 183: 1269-80). Importantly, the mesangial filopodia in
the GBM are depositing mesangial matrix proteins, including laminin
211, which activates focal adhesion kinase in glomerular podocytes,
resulting in the activation of genes encoding pro-inflammatory
cytokines and metalloproteinases, which drive the progression of
GBM damage. This is shown in Example 4 (see also Delimont et al.,
2014, PLoS One; 9(6):e99083). Thus, the mechanism underlying the
activation of mesangial filopodia will reveal novel targets of
therapeutic intervention aiming to arrest the initiation of these
events.
[0231] To explore this potential mechanism, C57Bl/6 X-linked Alport
mice were treated with L-NAME salts from 4 weeks to 7 weeks of age
to establish conditions of hypertension to compare with
normotensive mice. At 7 weeks this model is still pre-proteinuric.
Blood pressure was monitored thrice weekly using the CODA2 tail
cuff system ((Meehan et al., 2009, Kidney Int; 76:968-976). The
readings shown in FIG. 26 demonstrate that these treatments
resulted in blood pressure that varied by 15 to 25 mm of mercury
for both systolic and diastolic measurements.
[0232] It has been demonstrated that endothelin A receptor
activation on mesangial cells leads to RAC1/CDC42 activation
(Chahdi et al., 2005, J Biol Chem; 280(1):578-84; and Chahdi and
Sorokin, 2006, Exp Biol Med (Maywood); 231(6):761-5). Endothelin-1
expression was examined in cryosections from 7 week old
normotensive and hypertensive mice by immunofluorescence. FIG. 27
shows that under normotensive conditions, endothelin-1
immunolabeling in Alport glomeruli is weak, but more intense than
in glomeruli from wild type mice (compare panels A-C with panels
G-I). Under hypertensive conditions, immunostaining intensity in
Alport glomeruli is much greater (FIG. 27 (J-L)). Co-localization
with CD31 (an endothelial cell marker) demonstrates that the
endothelin immunostaining is coming from the endothelial cell
compartment. Similar differences in blood pressure in age/strain
matched wild type mice did not affect endothelin-1 immunostaining
intensity, which was of very low abundance (FIG. 27 (A-F)). Thus it
is apparent that strain mediated induction of endothelin-1 might be
responsible for inducing the formation of mesangial filopodia by
way of endothelin receptor activation. FIG. 28 shows western blot
analysis of glomerular lysates from wild type and Alport mice,
confirming that Alport mice express higher levels of
endothelin-1.
[0233] There are two classes of endothelin receptor; the endothelin
A receptor (ET.sub.AR) and the endothelin B receptor (ET.sub.BR).
Previous studies suggest that the primary endothelin receptor on
glomerular mesangial cells is the endothelin A receptor (Wendel et
al., 2006, J Histochem Cytochem; 54(11): 1193-203). This same study
showed that endothelin B receptors are primarily found on
glomerular endothelial cells and podocytes. To determine which
receptors are expressed on mouse mesangial cells,
immunofluorescence and western blot analysis were performed on both
glomeruli and cultured primary mesangial cells and cultured
podocytes. FIG. 29 (A) shows that the ET.sub.AR co-localizes with
the mesangial cell marker integrin .alpha.8, while the ET.sub.BR
localizes primarily to glomerular podocytes, which are identified
by the podocyte marker .alpha.-actinin-4. Western blots (FIG. 29
(B)) confirm that glomeruli express both ET.sub.AR and ET.sub.BR,
and cultured mesangial cells express only ET.sub.AR. Cultured
podocytes express both ET.sub.AR and ET.sub.BR, however ET.sub.BR
are expressed at higher levels.
[0234] While the link between endothelin treatment of cultured
mesangial cells and the activation of Rac1/CDC42 Rho GTPases has
been demonstrated, the connection between ET.sub.AR activation and
filopodia formation in mesangial cells has not. To address this,
serum-starved cultured mesangial cells were pre-treated (or not)
with the ET.sub.AR antagonist, Sitaxentan, and then stimulated the
cells with endothelin-1. Cells were then dual labeled with
anti-drebrin antibodies (drebrin stabilizes actin filaments in
filopodia) and phalloidin. The results in FIG. 30 (A) demonstrate
that treatment of cultured mesangial cells with endothelin-1
induces the formation of drebrin-positive filopodial microspikes on
cultured mesangial cells, and pretreatment of cells with the ETAR
antagonist Sitaxentan blocks the formation of microspikes. Lysates
from cultured mesangial cells were further analysed using these
same conditions for the activation of CDC42 using a commercial
ELISA assay for GTP-CDC42.
[0235] The results in FIG. 30 (B) show that endothelin-1 treatment
significantly activates CDC42 in these cells, and that pretreatment
with Sitaxentan prevents this activation. Combined these data
provide the scientific platform for in vitro studies proposed in
Aim 2.
[0236] To determine whether ETR antagonism prevents mesangial
filopodia formation in vivo, we treated 129 Sv Alport mice with
either Bosentan (ET.sub.AR and ET.sub.BR antagonist) or Sitaxentan
(an ET.sub.AR antagonist) from 2 weeks to 7 weeks of age. The data
from both inhibitors was essentially identical, which is consistent
with the biological effect (CDC42 activation and mesangial
filopodia formation) being due to ET.sub.AR signal transduction. In
the interest of space, we are providing the evidence generated
using the ET.sub.AR inhibitor, Sitaxentan. Both of these compounds
have been used clinically to treat pulmonary hypertension.
[0237] 129 Sv autosomal Alport mice were given Sitaxentan once
daily by oral gavage from 2 weeks to 7 weeks of age. Kidney
cryosections were immunostained using antibodies for laminin
.alpha.2 and integrin .alpha.8 to determine the effect of drug
treatment on laminin 211 deposition in the GBM or laminin .alpha.5
and integrin .alpha.8 to determine the degree of mesangial
filopodial invasion in the glomerular capillaries. In addition,
transmission electron microscopy was used to determine whether the
ET.sub.AR antagonist ameliorates GBM damage. The results in FIG. 31
demonstrate that vehicle-treated Alport mice showed extensive
invasion of glomerular capillaries by mesangial filopodia, which is
typical for this model at 7 weeks of age. Sitaxentan-treated mice
showed a near complete absence of mesangial filopodial invasion of
glomerular capillaries, looking much more like the wild type
glomeruli, especially with regard to the normalization of linear
laminin .alpha.5 immunostaining, rather than the irregular laminin
.alpha.5 immunostaining observed in glomeruli from vehicle-treated
mice. A blow-up of a vehicle treated Alport glomerulus
immunolabeled with laminin .alpha.5 and integrin .alpha.8 is
provided in FIG. 32 (A) which demonstrates that the often punctate
GBM integrin .alpha.8 immunolabeling is observed in all of the
capillary loops, consistent with filopodial invasion. FIG. 32B
shows quantitative analysis of total red fluorescence (integrin 8
immunostaining) in the capillary regions (laminin .alpha.5
immunopositive, but excluding the mesangial angles) from at least 6
independent glomeruli from at least three individual mice per
group. The data shows a significant elevation in capillary red
fluorescence in the glomeruli from vehicle-treated Alport mice
compared wild type mice, and normalization of capillary integrin 8
immunostaining in the Sitaxentan treated mice. Transmission
electron microscopic analysis of the GBM, which is shown at the
bottom of FIG. 31, demonstrates that Sitaxentan treatment resulted
in normalization of the GBM architecture.
[0238] Sitaxentan treated mice showed delayed onset of proteinuria,
as well as significantly reduced proteinuria after the onset (FIG.
33). Normally detected by 3 weeks of age, proteinuria was not
detected until 6 weeks of age in the treated mice. This is
consistent with the idea that endothelin receptor blockade works at
the level of disease initiation. Sitaxentan treatment also
profoundly ameliorated interstitial fibrosis and monocyte
recruitment to the renal interstitium, as evidenced by fibronectin
and CD11b immunostaining (FIG. 34). Very similar results were again
observed when animals were treated with Bosentan. Glomerular
expression of MMP-10, and -12 as well as the proinflammatory
cytokines TGF-.beta.1 and MCP-1 were significantly reduced in
Bosentan-treated Alport mice relative to Alport mice given vehicle
(FIG. 35). With the exception of TGF-.beta.1, Sitaxentan gave
quantitatively similar results. Collectively, these data indicate
that endothelin A receptor blockade is a novel therapeutic option
for the treatment of Alport syndrome, and works by blocking the
activation of RAC1 and CDC42, preventing the activation of
mesangial actin dynamics and thereby preventing the invasion of
glomerular capillaries by mesangial filopodia. This represents a
previously unrecognized etiology for the action of endothelin
receptor blockade in the treatment of biomechanical strain-mediated
Alport glomerular disease initiation.
[0239] The data presented above provide strong evidence that
endothelin receptor activation results in mesangial process
invasion of glomerular capillaries, indicating that additional drug
targets aimed at blocking Alport glomerular disease initiation by
uncoupling strain-mediated induction of endothelin-1.
Specific Aim 1
[0240] Cyclic cell stretching glomerular endothelial cells will
result in elevated expression of endothelin-1 and other known
stretch-responsive genes via the TRPC3/zyxin pathway.
[0241] Previously published work shows that mechanical stretching
of umbilical cord-derived human endothelial cells, and aorta
derived murine endothelial cells results in elevated expression of
endothelin-1 (Wojtowicz et al., 2010, Circ Res; 107(7): 898-902;
Babu et al., 2012, Sci Signal; 5(254):ra91), a finding also
observed in mechanically stretched astrocyte cultures (Ostrow et
al., 2011, Biochem Biophys Res Commun; 410(1):81-6). Similar
observations have not yet been documented for glomerular
endothelial cell cultures. We have derived and qualified
conditionally immortalized glomerular endothelial cells from
glomerular outgrowths of the immortomouse. Our data illustrates
that endothelin-1 protein expression is elevated in the endothelial
cell compartment or hypertensive pre-proteinuric Alport mice
relative to normotensive Alport mice (FIG. 27), suggesting that
biomechanical stretching of glomerular endothelial cells induces
endothelin-1 gene expression. Since glomerular endothelial cells
are distinct from endothelial cells from the aorta or the umbilical
veins, we will perform experiments to confirm that the pathway for
stretch-induced gene expression is intact in our conditionally
immortalized glomerular endothelial cell culture system. Based on
preliminary evaluation of our conditionally immortalized cell
cultures by real time RT-PCR, we know that they express the
molecular machinery for this induction pathway (including TRPC3,
ET-1, ER.sub.BR, GC-A, protein kinase G and zyxin).
[0242] Cells will be differentiated for 2 weeks, serum starved, and
plated on 6-well Flexcell plates pre-coated with rat tail collagen.
Biomechanical stretching will be applied overnight using the
Flexcell tension system (Flexcell International Corporation) as
described in Example 1 (see also Meehan et al., 2009, Kidney Int;
76:968-976); and Zallocchi et al., 2013, Am J Pathol;
183(4):1269-80). RNA from these cells as well as cells treated
identically but not subjected to biomechanical stretching will be
analyzed by real time RT-PCR for expression of endothelin-1, Hey-1,
VCAM-1 and IL-8 (all transcripts shown to be induced by stretching
umbilical cord-derived primary human endothelial cells (HUVEC);
(Wojtowicz et al., 2010, Circ Res; 107(7): 898-902). Primary HUVEC
cell cultures will be used as a positive control in all experiments
to assure that the conditions are conducive to induction of this
gene set, and to determine whether the inhibitors, as applied in
our system block induction of these same genes, as was previously
described (Babu et al., 2012, Sci Signal; 5(254):ra91). Inhibitors
for TRPC3 (Pyr3) and protein kinase G (Rp8) will be applied prior
to stretching to assess the effect of these inhibitors on induction
of the gene set. Protein kinase G phosphorylates zyxin, allowing it
to dissociate from the plasma membrane focal adhesions and
translocate to the nucleus where it activates expression of the
zyxin responsive genes (Babu et al., 2012, Sci Signal;
5(254):ra91). In addition to the real time RT-PCR analysis, we will
immunostain stretched and mock-stretched cells for all conditions
with anti-zyxin antibodies to determine whether zyxin has
translocated to the nucleus, consistent with its activation.
[0243] Preproteinuric X-linked Alport mice have very little GBM
damage as determined by transmission electron microscopy, which
predicts a thinner and less crosslinked GBM would result in the
capillary tufts being more susceptible to biomechanical stretching
than that of age/strain-matched wild type mice. Given the fact that
pre-proteinuric Alport mice have elevated levels of endothelin-1
relative to age/strain-matched wild type mice (FIGS. 27-28), and
that hypertensive Alport mice show further elevation of
endothelin-1 expression, specifically in the endothelial cell
compartment of the glomerulus (FIG. 27), we predict that the
biomechanical stretching of cultured glomerular endothelial cells
will result in the induction of the endothelin-1, Hey-1, VCAM-1 and
IL-8 transcripts, as was observed and reported for both HUVECs and
murine aortic endothelial cells (Wojtowicz et al., 2010, Circ Res;
107(7): 898-902; Babu et al., 2012, Sci Signal; 5(254):ra91). We
also feel that is highly likely that the TRPC3/zyxin pathway is
intact in these cells, since our conditionally immortalized
glomerular endothelial cells express all of the requisite molecules
and since we do not observe elevated expression of endothelin-1 in
zyxin/COL4A5 double knockout mice relative to age/strain-matched
wild type mice. It is likewise expected that the pre-incubation of
cells with TRPC3 or protein kinase G inhibitors will, upon cyclic
stretching, block the translocation of zyxin to the nucleus and
block the induction of zyxin-responsive genes.
Specific Aim 2
[0244] Newly identified regulators are required for endothelin-1
mediated activation of CDC42 and subsequent formation of
drebrin-positive actin microspikes in cultured mesangial cells.
RNAseq analysis was performed on glomerular RNA from C57 Bl/6 wild
type and X-linked Alport animals. 7 week-old animals were chosen
because these animals are preproteinuric indicating a functionally
intact GBM. We found that a number of genes were modulated (either
up or down) in the Alport glomeruli relative to wild type. At least
five of these genes, which were significantly induced in Alport
glomeruli relative to wild type, are functionally linked to the
activation pathway for CDC42. These include BMP-7 (bone
morphogenetic protein-7) and its receptor BMP-RII, both of which
have previously been shown to be expressed by mesangial cells (Yeh
et al, 2009, Biochem Biophys Res Commun; 382(2):292-7), and four
genes that are known to be implicated in the activation of CDC42 in
other systems, but have never been described in the glomerulus or
mesangial cells, including T cell differentiation protein 2 (MAL2)
(Madrid et al., 2010, Dev Cell; 18(5): 814-27), golgi autoantigen,
golgin subfamily a,2 (GM130) (Kodani et al., 2009, Mol Biol Cell;
20(4): 1192-200), wingless-related MMTV integration site 11 (Wntl
1) (Choe et al., 2013, Dev Cell; 24(3):296-309), and
sortillin-related VPS10 domain containing receptor 2 (sorcs2)
(Deinhardt et al., 2011, Sci Signal; 4(202):ra82). Interestingly,
MAL2 and GM130 are involved in the trafficking of CDC42 to the
plasma membrane, which is required for its activation (Madrid et
al., 2010, Dev Cell; 18(5): 814-27; Kodani et al., 2009, Mol Biol
Cell; 20(4):1192-200; Osmani et al., 2010, J Cell Biol; 191(7):
1261-9). Real-time RT-PCR was performed using RNA from primary
cultured mesangial cells to determine whether these newly
identified glomerular transcripts are expressed in the mesangial
cell compartment. The results demonstrated that all 6 transcripts
linked to CDC42 activation and induced in Alport glomeruli from
pre-proteinuric mice are abundantly (CT<30) expressed in the
cultured mesangial cells.
[0245] The development of the in vitro bioassay for
endothelin-mediated CD42 activation (FIG. 30) provides the platform
for determining whether these genes are indeed linked to CDC42
activation in cultured mesangial cells. Transient SiRNA knockdown
studies will be undertaken for each of the six newly identified
genes. Following transfection with either gene-specific SiRNA or
scrambled SiRNA, the cells will be incubated 24-48 hours, and then
serum starved. Cells will then be stimulated with endothelin-1 and
evaluate the cultures for the presence of cells with
drebrin-positive actin-rich microspikes by dual staining with
phalloidin and anti-drebrin antibodies. The cultures will be
further evaluated for activated CDC42 using a commercially
available ELISA assay as in FIG. 30. ELISA will be used as opposed
to pull-down assays because the amount of material required for the
latter is large, making this approach impractical. For the MAL2 and
GM130 siRNA knockdowns (these proteins are implicated in the
trafficking of CDC42 to the plasma membrane, which is a
pre-requisite for activation, immunostaining with anti-CDC42
antibodies will determine whether the CDC42 is associated with the
plasma membrane. As a control, we will knock down expression of the
guanine nucleotide exchange factor, p21-activated
kinase-interacting exchange factor (.beta.1Pix), which has
previously been shown required for the activation and membrane
localization of CDC42.
[0246] Given that all 6 genes to be analyzed are induced very early
in Alport glomerular pathogenesis, and that all of them are
relatively abundant in cultured mesangial cells (CT values of 30 or
less by real time RT-PCR), we expect them to be functionally
important. Given the documentation in other cell systems that the
proteins encoded by these genes are implicated in CDC42 activation,
it is likely they play a role in this pathway in mesangial cells.
Therefore we expect that we will find evidence that all or most of
these proteins are indeed important in the activation of CDC42,
identifying new aspects of this pathway that can be further
explored in the mechanism of mesangial cell adhesion, migration,
and filopodial formation, which has broader implications in the
field of glomerular disease in addition to its importance in
understanding the activation of filopodia formation in the onset of
Alport glomerular disease.
Specific Aim 3
[0247] An endothelial cell-specific endothelin knockout Alport
mouse will show arrested mesangial process invasion of the
glomerular capillaries, preventing laminin 211-mediated FAK
activation and ameliorating the initiation/progression of
glomerular pathology. In the results discussed herein, we show that
endothelin-1 is induced in glomeruli from pre-proteinuric Alport
mice (FIGS. 27-28), but not wild type mice. We also show that
mesangial cells express the endothelin A receptor, but not the
endothelin B receptor (at least not within the detection limits of
the methods used). As discussed in the previous example, Endothelin
B receptors are the principal endothelin receptors found on
glomerular endothelial cells and podocytes (see also Wendel et al.,
2006, J Histochem Cytochem; 54(11):1193-203; Yamamoto et al., 2002,
Arch Histol Cytol; 65(3):245-50). Blockade with endothelin A
receptor antagonists was as effective as Bosentan at ameliorating
glomerular and interstitial disease, which blocks both receptors,
suggesting that the activation of actin cytoskeletal dynamics in
the mesangial cell compartment occurs as a result of endothelin A
receptor activation. Treatment of pre-proteinuric Alport mice with
endothelin receptor blocking agents significantly reduced mesangial
filopodial invasion of the glomerular capillaries, deposition of
laminin 211 in the GBM, elevated expression of pro-pathogenic gene
expression, delayed the onset and progression of proteinuria, and
ameliorated the GBM dysmorphology (FIGS. 31-35).
[0248] As shown in FIG. 27, endothelin-1 is by far predominantly
expressed in the glomerular endothelial cells. Earlier studies
demonstrate that endothelin-1 secretion is highly regulated in
glomerular endothelial cells, suggesting important homeostatic
functions (Marsden et al., 1991; Babu et al., 2012, Sci Signal;
5(254):ra91). It has been speculated that endothelin-1 secreted by
glomerular endothelial cells influences mesangial cell
contractility and function (Simonson and Dunn, 1990, J Clin Invest;
85(3):790-7). Endothelin-1 knockout mice are not viable due to
defects in the development of the heart and great vessels (Kurihara
et al., 1995, J Clin Invest; 96(1):293-300). Interestingly, over
expression of endothelin-1 causes late onset glomerulosclerosis,
demonstrating that levels of endothelin-1 in healthy glomeruli are
likely under tight homeostatic regulatory control (Hocher et al.,
1997, J Clin Invest; 99(6): 1380-9).
[0249] A direct way to test the role of endothelin-1 in mesangial
process invasion of glomerular capillaries is to remove
endothelin-1 expression from the system by way of genetic modeling.
Given the lethality of the global knockout for EDN1, we will employ
an endothelial cell-specific conditional knockout approach. This
approach has been previously developed using the Tie2-Cre
transgenic to drive endothelial cell-specific deletion of the
floxed EDN1 gene (Kisanuki et al., 2010, Hypertension;
56(1):121-8). Importantly, these mice are found to be viable with
no noted abnormalities, and have normal life spans, but
interestingly lower blood pressure. This same paper documented
complete deletion of endothelin-1 immunostaining in the glomerular
endothelial cells of the Tie2-Cre (+) EDN flox/flox mouse,
providing proof of concept that this system will work for our
purposes. The EDN1flox/flox mouse is also on the C57 Bl/6
background. These transgenics will be bred with the C57Bl/6
X-linked Alport knockout mouse to produce the endothelial
cell-specific EDN1 knockout Alport mouse.
[0250] This approach will allow us to determine the extent of
improved renal function that can be achieved by blocking endothelin
receptor activation in Alport mesangial cells. In addition, by
comparing hypertensive and hypotensive conditional EDN1 Alport mice
we will be able to ascertain whether biomechanical strain results
in other effects that promote Alport glomerular pathology that are
independent of endothelin-1 mediated activation of glomerular
mesangial cells. For instance, it has been shown that biomechanical
stretching of glomerular podocytes activates angiotensin II
receptors and increases expression of secreted protein acidic and
rich in cysteine (SPARC) (see, for example, Durvasula and
Shankland, 2005, Am J Physiol Renal Physiol; 289(3):F577-84). The
former is associated with apoptosis, and the latter is associated
with accelerated renal injury in diabetic mice. Thus, biomechanical
stretching of podocytes has been shown to have some direct
maladaptive effects on podocyte biology both in vivo and in
vitro.
[0251] The experimental design strategy will consist of two
different experiments. In the first we will compare the disease
progression in wild type, EDN1 conditional KO, Alport, and EDN1
conditional KO Alport mice. All mice to be used in the study will
be derived from the Tie2-Cre/Alport/EDN1 flox/flox intercrosses.
The first experiment will consist of one set of mice to be used for
collection of time points, which will be collected at 5 week
intervals starting at 5 weeks of age until 20 weeks of age (the
mean lifespan for the X-linked Alport mouse model on this
background is 25 weeks), and a second set of mice that will be used
for longitudinal assessment of proteinuria, blood urea nitrogen
levels, and lifespan. For the first set of mice, one kidney will be
used for immunohistochemical analysis and transmission electron
microscopy (TEM), and the other kidney will be used for RNA
isolation by perfusion of glomeruli with magnetic Dynabeads.
Glomerular RNA will be analyzed by real time RT-PCR for transcripts
indicative of glomerular disease progression based on our earlier
studies. These are essentially used as biomarkers since, as shown
in Example 6, they are significantly induced in murine Alport
glomeruli (see also Delimont et al., 2014, PLoS One; 9(6):e99083).
These include MMP-10, MMP-12, MCP-1, and TGF-.beta.1. Based on our
experience with the mouse X-linked Alport mouse model, 5 animals
per group will provide enough power to determine whether
statistically significant differences exist in the expression of
these genes when comparing groups. The five week intervals will
provide information regarding the temporal kinetics of the
therapeutic effect of EDN1 deletion. TEM analysis will provide an
indication as to whether the EDN1 deletion improves the progression
of the GBM damage and dysmorphology. Dual immunohistochemical
staining for laminin .alpha.2/pFAK.sup.397, laminin
.alpha.2/laminin .alpha.5, and integrin .alpha.8/laminin .alpha.5
will provide evidence regarding the degree to which endothelial
cell specific EDN1 deletion blocks mesangial filopodial invasion of
the glomerular capillary loops, laminin 211 deposition in the GBM,
and maladaptive pFAK activation in glomerular podocytes as a
function of disease progression. The second experimental group will
provide information on glomerular function (BUN/proteinuria) and
will be conducted using methods are standardized in the laboratory.
Lifespan will also be assessed, which will provide an indication as
to the extent to which endothelin-1 receptor blockade might improve
glomerular disease progression when fully optimized.
[0252] The second set of experiments are to define whether the
effects of biomechanical strain are truly rooted in induction of
endothelin-1 in glomerular endothelial cells (as shown in FIG. 8)
and its effects on endothelin A receptor mediated activation of
mesangial filopodial invasion of the glomerular capillaries (As
shown in FIG. 34). Previously published work shows that we can
markedly accelerate glomerular disease progression in Alport mice
by making them hypertensive. These studies were the foundation of
the hypothesis that the biomechanical properties of Alport GBM
likely played a role in the mechanisms underlying glomerular
pathogenesis. The conditional EDN1 knockout Alport mouse has a
basement membrane collagen network comprised only of
.alpha.1(IV)/.alpha.2(IV) chains, and thus the glomerular
capillaries in pre-proteinuric mice are likely constantly subjected
to abnormally high biomechanical stresses (a chronic insult that
drives initiation and progression). The purpose of this experiment
is to identify whether additional strain-dependent events occur
that lie outside of the endothelin-1-mediated mesangial cell
activation axis. Five animals per group as described above will be
given L-NAME salts or regular water from 4 weeks until 10 weeks of
age and blood pressure monitored once weekly using the CODA 2 tail
cuff system. These ages are chosen based on our previously
published findings, which demonstrate a clear influence of
hypertension on glomerular disease progression within this time
interval in the C57Bl/6 X-linked model (Meehan et al., 2009, Kidney
Int; 76:968-976)). Urine will be collected at weekly intervals for
proteinuria determination and blood will be collected at the
termination of the experiment (10 weeks) for BUN measurements. One
kidney will be used for RNA isolation and the other kidney for
immunohistochemical analysis and TEM as for the first set of
experiments. Analysis will be conducted as per above, with the
exception that mRNA and immunohistochemistry will be conducted for
SPARC expression, as this podocyte marker proved to be a reliable
indicator of a biomechanical strain-mediated maladaptive
podocyte-specific response in earlier work (Durvasula and
Shankland, 2005, Am J Physiol Renal Physiol; 289(3):F577-84).
[0253] As shown in FIG. 27, endothelin A receptors are only
detected on mesangial cells in the glomerulus (see also Wendel et
al., 2006, J Histochem Cytochem; 54(11):1193-203), and the
endothelin A receptor-specific antagonist Sitaxsenten ameliorates
Alport glomerular disease progression at least as well as Bosentan,
which blocks both endothelin A and B receptors. Based on these
facts, we are confident that mesangial filopodial invasion results
from paracrine activation of endothelin A receptors on mesangial
cells by endothelial cell-derived endothelin-1. Endothelial
cell-specific deletion of endothelin-1 should therefore produce a
renoprotective phenotype similar to the endothelin A receptor
blocking agent, Sitaxsentan. Since biomechanical stretching
elevates expression of glomerular endothelial cell-derived
endothelin-1, we predict that salt-mediated hypertension will not
accelerate glomerular disease progression in the conditional EDN1
KO Alport mouse, but will accelerate progression in the Alport
mouse. It is possible that hypertension will accelerate disease
progression in the EDN1 conditional KO Alport mouse. If we observe
this, along with elevated SPARC expression in the podocytes of
these mice, we will interpret that to mean that stretch-mediated
influences in the podocyte compartment may be a significant
contributor to progression of Alport glomerular disease and explore
this mechanism further.
Specific Aim 4
[0254] Dislocation of strain mediated induction of endothelin-1 by
way of either TRPC3 or zyxin knockout will arrest endothelin
induction and ameliorate mesangial process invasion of the
glomerular capillaries, preventing laminin 211-mediated FAK
activation and ameliorating the initiation/progression of Alport
glomerular pathology, defining new targets for therapeutic
intervention. Bosentan (endothelin A and B receptor blocker) and
Ambrisentan (Endothelin A receptor-specific blocker, US trade name,
LETAIRIS) are FDA approved drugs for the treatment of pulmonary
hypertension. Both receptor blocking strategies have proven highly
effective at reducing glomerular pathology and interstitial
fibrosis in the Alport mouse model, as shown in the results
described herein (we used Sitaxsenten as the endothelin A blocker
because Ambrisentan was not available). Both were highly effective
at ameliorating mesangial filopodial invasion and deposition of
laminin 211 in the glomerular capillaries, and at reducing
maladaptive regulation of MMPs and pro-inflammatory cytokines.
These properties predict that endothelin receptor blockade (more
specifically endothelin A receptor-specific blockade) may prove to
be a promising new therapeutic approach for the treatment of Alport
renal disease. One potential problem with this approach is that
these drugs have a number of side effects. While acceptable for
adults and children suffering from pulmonary hypertension, these
side effects may preclude their use in children with Alport
syndrome, since the therapy would continue in these patients for
decades.
[0255] The purpose of this aim is to uncouple the mechanism of
endothelin induction in glomerular endothelial cells using genetic
modeling in order to reveal new potential targets for pharmacologic
intervention. Biomechanical strain has been shown to induce the
production and release of endothelin-1 in both astrocytes and
endothelial cells (Hishikawa et al., 1995, Hypertension;
25(3):449-52; Ostrow et al., 2000, J Cardiovasc Pharmacol; 36(5
Suppl 1):S274-7). In endothelial cells, a major regulator of the
mechanotransduction apparatus was found to be the LIM domain
containing protein zyxin (Cattaruzza et al., 2004, Circ Res;
95(8):841-7). The zyxin protein is normally found at focal
adhesions and translocates to the nucleus following a
stretch-mediated phosphorylation event, where it acts as a
transcription factor up-regulating pro-inflammatory gene
expression, including endothelin-1 (Wojtowicz et al., 2010, Circ
Res; 107(7): 898-902). Recently it was shown that stretch-mediated
activation of the transient receptor potential channel 3 (TRPC3)
triggers a signaling cascade in endothelial cells culminating in
the phosphorylation of zyxin, resulting in its release from focal
adhesions and translocation to the nucleus (Babu et al., 2012, Sci
Signal; 5(254):ra91). The authors demonstrated that this signaling
cascade can be uncoupled at various points in vitro to block zyxin
activation, and thus block stretch-mediated responses in
endothelial cells.
[0256] The present approach will produce double knockout mice for
the most upstream (TRPC3 activation) and downstream (zyxin
activation) events in this cascade. Both zyxin and TRPC3 knockout
mice are available from national repositories (MMRRC and Jackson
Laboratories, respectively). Both of these knockout mice are
fertile and have normal life spans. The TRPC3 mice show impaired
walking behavior, presumably due to neurological effects. The zyxin
knockout mice are on a pure C57Bl/6 background, and therefore can
be readily bred to the X-linked Alport mouse model on this same
background. The TRPC3 knockout mice are on a mixed 129 Sv/C57 Bl/6
background and thus will need to be backcrossed to C57 Bl/6 before
a double knockout can be produced. This is critical as we know that
strain-associated genetic modifiers exist that can profoundly
affect renal disease progression which would surely introduce an
unacceptable degree of variability on a mixed background.
[0257] The induction of endothelin-1 that we demonstrated in the
endothelial cell compartment of hypertensive Alport mice relative
to normotensive or hypotensive Alport mice (FIG. 27, N=4) combined
with the demonstrated role for TRPC3 mediated activation of zyxin
in this process would predict that endothelin mRNA and protein
expression levels would be absent or normalized to basal
(non-strain regulated) levels in both zyxin null and TRPC3 null
Alport mice. As mentioned, we have already produced the
zyxin/COL4A5 double knockout mouse model, and observed very low
levels of endothelin-1 expression in this model, consistent with
our earlier assumptions, and providing strong support for this
approach. All experimental animals will be derived from
intercrosses for each double knockout mouse model. We will examine
wild type, Alport, TRPC3 or zyxin KO, and TRPC3 KO/Alport or zyxin
KO Alport mice. Animals will be treated with Ramipril or L-NAME
salts from 4 weeks until 10 weeks of age with blood pressures
monitored once a week. Glomeruli will be isolated from one kidney
for analysis of endothelin mRNA and protein by real time RT-PCR and
western blot. Expression levels of MMP-10, MMP-12, and MCP-1 RNA
will also be examined (based on preliminary data with endothelin
receptor blockers). The other kidney will be examined by TEM for
GBM dysmorphology and by immunohistochemistry for GBM laminin 211
deposition, podocyte FAK activation (pFAK397 immunostaining) and
mesangial interposition by integrin 8 immunostaining.
[0258] A second set of experiments will be conducted specifically
as outlined for Aim 1, where we will examine proteinuria, BUN and
lifespan on one set of animals and the kinetics of disease
progression at 5, 10, 15 and 20 weeks of age in the second set of
animals.
[0259] Based on the published findings linking stretch mediated
TRPC3 activation to zyxin activation in endothelial cell
mechanotransduction (Babu et al., 2012, Sci Signal; 5(254):ra91) we
feel confident that the gene deletions will ameliorate glomerular
disease progression in the Alport mouse model. The fact that we
observe elevated expression of endothelin-1 in the endothelial
cells of Alport mice as well as the observation that hypertension
further elevates the expression of endothelin-1 infers that this
strain-regulated pathway is activated in our model. If true,
deletion of either TRPC3 or zyxin should short circuit this
signaling pathway resulting in the absence of elevated endothelin-1
expression in the glomerular endothelial cells and the amelioration
of endothelin-mediated influences, including abrogation of
mesangial filopodial invasion of the glomerular capillaries,
deposition of laminin 211 in the GBM, and activation of FAK in
Alport podocytes. To date, we have confirmed that 7 week old
(preproteinuric) zyxin/COL4A5 double knockout mice express low
levels of endothelin-1 relative to age/strain matched COL4A5 null
mice, consistent with our central hypothesis. In the work described
by Wojtowicz et al. (Wojtowicz et al., 2010, Circ Res; 107(7):
898-902), global gene expression analysis of stretched endothelial
cells demonstrated that stretch mainly activated genes involved in
pro-inflammatory pathways. Based on this, we might expect that
these double knockout mice could be more effective at preventing
the onset and/or progression of glomerular disease than the
conditional knockout of endothelin-1 (Aim 3) or endothelin blockade
with small molecule inhibitors (preliminary results). If this
scenario is observed, it would suggest that therapeutic approaches
aimed at this strain-responsive signaling cascade might provide
superior renoprotection in Alport patients compared to endothelin
blockade alone, warranting the development of new drugs that target
this pathway. It is notable that endothelin receptor blockade by
either Bosentan or Ambrisentan, while FDA approved for pulmonary
fibrosis, might be considered toxic for use in young boys with
Alport syndrome. This fact provides justification for exploring
alternative means of targeting the strain-mediated regulation of
endothelin-1 in Alport syndrome.
Example 7
Neutralizing Antibodies to Endothelin-1
[0260] Following procedures described in the previous examples, the
effect of neutralizing antibodies to endothelin-1 or the endothelin
receptor will be tested for their ability to prevent mesangial
filopodial invasion of glomerular capillaries and delay Alport
glomerular and interstitial disease onset.
[0261] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for instance, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PDB, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference. In
the event that any inconsistency exists between the disclosure of
the present application and the disclosure(s) of any document
incorporated herein by reference, the disclosure of the present
application shall govern. The foregoing detailed description and
examples have been given for clarity of understanding only. No
unnecessary limitations are to be understood therefrom. The
invention is not limited to the exact details shown and described,
for variations obvious to one skilled in the art will be included
within the invention defined by the claims.
* * * * *